Compostions and methods for identifying response targets and treating flavivirus infection responses

ABSTRACT

Cellular receptors are identified that induce plasma leakage and other negative effects when infected with flaviviruses, such as dengue virus or Japanese encephamyelitis virus. Using fusion proteins disclosed herein, the receptors to which a pathogen, such as flavivirus, binds via glycan binding are determined. Once the receptors are determined, the effect of binding to a particular receptor may be determined, wherein targeting of the receptors causing a particular symptom may be targeted by agents that interrupt binding of the pathogen to the receptor. Accordingly, in the case of dengue virus and Japanese encephamyelitis virus, TNF-α is released when the pathogen binds to the DLVR1/CLEC5A receptor. Interrupting the DLVR1/CLEC5A receptor with monoclonal antibodies reduced TNF-α secretion without affecting secretion of cytokines responsible for viral clearance thereby increasing survival rates in infected mice from nil to around 50%.

This application claims the Paris Convention priority and is acontinuation-in-part of U.S. Utility patent application Ser. No.11/469,270, filed Aug. 31, 2006, which claims the Paris Conventionpriority of U.S. Provisional Patent Application Ser. No. 60/713,463,filed Aug. 31, 2005, the disclosures of which are incorporated herein byreference in their entirety.

This work was supported by grant 94F008-5, NSC 95-2320-B-010-010 and NSC95-3112-B-010-017 from the National Sciences Council, Taiwan. This workwas also supported by grant 94M002-1 from the Academia Sinica, Taiwan,and by grant 95A-CT8G02 from the National Yang-Ming University.

BACKGROUND

Citation to any reference in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that thisreference forms part of the common general knowledge or of the prior artin any country. All references cited herein are specificallyincorporated herein by reference in their entirety.

The immune system enables a host organism to discriminate self fromnon-self antigens, as well as to recognize and eradicate invasivepathogens. The adaptive immunity system relies on highly polymorphicmolecules, such as class I and class II antigens of the majorhistocompatibility complex (MHC), T cell receptors, and B cellreceptors, to present antigens to T cells and B cells, thus leading tothe activation of immune system. The mechanism by which the innateimmunity system can recognize these diverse antigens remained unsolveduntil the emergence of the concept of ‘pattern recognition receptors(PPRs)’ proposed by Janeway (Janeway, 1989, Cold Spring Harb Symp QuantBiol 54 Pt 1, 1-13). This hypothesis was later proved correct by theidentification of pathogen-associated molecular patterns (PAMPs) whichare recognized by TOLL-like receptors (Aderem and Ulevitch, 2000 Nature406, 782-7; Akira and Takeda, 2004, Nat Rev Immunol 4, 499-511; Athmanand Philpott, 2004, Curr Opin Microbiol 7, 25-32), lectin receptors(Cambi and Figdor, 2003, Curr Opin Cell Biol 15, 539-46),immunoglobulin-like (Ig-like) receptors (Daws et al., 2003, J Immunol171, 594-9), and NOD proteins (Athman and Philpott, 2004, Curr OpinMicrobiol 7, 25-32), and others (Liu et al., 2001, J Biol Chem 276,34686-94; McDonald et al., 2005, J Biol Chem 280, 20177-80).

In addition to the well characterized PAMPs recognized by TOLL-likereceptors (Akira and Takeda, 2004, Nat Rev Immunol 4, 499-511), recentstudy indicates that the host immune system can recognize invasivepathogens through specific carbohydrate antigens. For example, mannosereceptors can recognize the high mannose sugar moiety expressed on thesurface of pathogens (Stahl and Ezekowitz, 1998, Curr Opin Immunol 10,50-5), while the Dectin-1 receptor can bind specifically to β-glucan,the major backbone of polysaccharides on fungus walls (Brown and Gordon,2001, Nature 413, 36-7; Herre et al., 2004, Mol Immunol 40, 869-76).These results suggest that the carbohydrate structures associated withpathogens are one of the targets recognized by the innate immunityreceptors of immune cells.

The funguses species Ganoderma and Cordyceps are the most popular herbaldrugs taken in China to medicinal purposes. Polysaccharides extractedfrom Ganoderma lucidum (also known as Ling zhi, Reishi) have been usedin traditional Chinese medicine as anti-tumor agents and asimmuno-modulating agents (Lien, 1990, Prog Drug Res 34, 395-420; Wang etal., 2002, Bioorg Med Chem 10, 1057-62; Shiao, 2003, Chem Rec 3,172-80), while those extracted from Cordyceps sinensis (Cordyceps,Caterpillar fungus) have been shown to alter apoptotic homeostasis, andto improve respiratory, renal, and cardiovascular functions (Buenz etal., 2005, J Ethnopharmacol 96, 19-29; Zhu et al., 1998, J AlternComplement Med 4, 289-303; Zhu et al., 1998, J Altern Complement Med 4,429-57), as well as to increase whole body sensitivity to insulin (Balonet al., 2002, J Altern Complement Med 8, 315-23). However, thepolysaccharide composition of the extracts vary when they thepolysaccharides are extracted from different sources, from differentstrains, and under different growing conditions.

Analytical methods relying on high-performance liquid chromatography(HPLC) and proton-nuclear magnetic resonance have been applied toinvestigate the components of polysaccharides isolated from Ganodermalucidum and Cordyceps sinensis (He and Seleen, 2004, Int. J. Med.Mushrooms 6, 253). However, the HPLC chromatogram is based on thecomparison with ganoderic acid A and C (two major triterpenes ofGanoderma lucidum) or adenosine. It is still difficult to know whetherthe extracts contain the active components of polysaccharides based onthe mass spectrum.

Cellular receptors are identified that induce plasma leakage and othernegative effects when infected with flaviviruses, such as dengue virusor Japanese encephamyelitis virus. Using fusion proteins disclosedherein, the receptors to which a pathogen, such as flavivirus, binds viaglycan binding are determined. Once the receptors are determined, theeffect of binding to a particular receptor may be determined, whereintargeting of the receptors causing a particular symptom may be targetedby agents that interrupt binding of the pathogen to the receptor.Accordingly, in the case of dengue virus and Japanese encephamyelitisvirus, TNF-α is released when the pathogen binds to the DLVR1/CLEC5Areceptor. Interrupting the DLVR1/CLEC5A receptor with monoclonalantibodies reduced TNF-α secretion without affecting secretion ofcytokines responsible for viral clearance thereby increasing survivalrates in infected mice from nil to around 50%.

SUMMARY

According to a feature of the present disclosure, a method is disclosedcomprising obtaining a complement of fusion proteins, each fusionprotein comprising a binding domain of a receptor and a domain thatprovides for affixing to a substrate, contacting the fusion protein witha pathogen to determine if the pathogen binds to the binding domain ofat least one fusion protein of the complement of fusion proteins, anddetecting whether the pathogen is bound to the fusion protein. Thecomplement of fusion proteins represents a plurality of differentbinding domains of at least one receptor.

According to a feature of the present disclosure, a method is disclosedcomprising obtaining cells susceptible to a pathogen, knocking down atleast one cellular receptor gene, contacting the cells with thepathogen, and measuring the level of cytokine secretion of the cells.

According to a feature of the present disclosure, a method is disclosedcomprising identifying at least one cellular receptor that binds to aligand displayed by a pathogen and administering an agent to an animalinfected with the pathogen to interrupt binding of the ligand to thereceptor to modulate an effect of the pathogen.

According to a feature of the present disclosure, a method is disclosedcomprising providing an effective amount of an agent to modulate theeffect of a pathogen infecting an animal to modulate the effect of thepathogen on the animal. The agent is directed to at least one cellularreceptor of the animals native cells to prevent the receptor frombinding to a ligand presented by the pathogen.

According to a feature of the present disclosure, a method is disclosedcomprising providing an effective amount of an anti-DLVR1/CLEC5Aantibody to an animal infected with dengue virus, wherein theanti-DLVR1/CLEC5A antibody prevents binding of a ligand presented by adengue virus particle from binding to the DLVR1/CLEC5A receptor, whereinsecretion of TNF-α is inhibited.

According to a feature of the present disclosure, a method is disclosedcomprising providing an effective amount of an anti-DLVR1/CLEC5Aantibody to an animal infected with Japanese encephamyelitis virus,wherein the anti-DLVR1/CLEC5A antibody prevents binding of a ligandpresented by a Japanese encephalitis particle from binding to theDLVR1/CLEC5A receptor, wherein secretion of TNF-α is inhibited.

According to a feature of the present disclosure, a method is disclosedcomprising providing to an animal infected with dengue virus aneffective amount of an agent that at least partially inhibits thesecretion of at least one pro-inflammatory cytokine without affectingthe secretion of interferon-α.

According to a feature of the present disclosure, a mouse is disclosedcomprising a mouse susceptible to dengue virus infection, and an sh-RNAparticle to knock down the DLVR1/CLEC5A receptor in the mouse.

According to a feature of the present disclosure, a composition isdisclosed comprising a pharmaceutical preparation containing aneffective amount of an antibody directed against at least one cellularreceptor of an animal to modulate the effects of a pathogen infection inthe animal. The modulation comprises at least inhibition inpro-inflammatory cytokine secretion of the animals cells and does notaffect secretion of cytokines that effect viral clearance.

According to a feature of the present disclosure, a composition isdisclosed comprising a pharmaceutical preparation containing aneffective amount of an antibody directed against the DLVR1/CLEC5Areceptor of an animal infected with dengue virus to modulate the effectsof the dengue virus infection in the animal. The modulation comprises atleast inhibition in pro-inflammatory cytokine secretion the animalscells and does not affect secretion of cytokines that effect viralclearance.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee. The above-mentioned features and objects of thepresent disclosure will become more apparent with reference to thefollowing description taken in conjunction with the accompanyingdrawings wherein like reference numerals denote like elements and inwhich:

FIG. 1A shows DNA fragments of innate immunity receptors amplified byRT-PCR, then fractionated on 0.8% agarose and visualized by ethidiumbromide staining. FIG. 1B shows the expressed recombinant receptor.Fcfusion proteins following electrophoresis on a 12% SDS-PAGE gel;

FIG. 2A shows a dot blot of membrane-immobilized biotinylated GLPS F3contacted with streptavidin-conjugated horseradish peroxidase (HRP).

FIG. 2B shows a dot blot of membrane-immobilized non-biotinylated GLPSF3 contacted with a Dectin-1.Fc fusion protein, followed by incubationwith goat HRP-conjugated anti IgG1 antibody. FIG. 2C shows a dot densityanalysis of the blot of FIG. 2B. FIG. 2D shows the effects on dotdensity of competitor β-glucan on the binding of Dectin-1.Fc tomembrane-immobilized GLPS F3. FIG. 2E shows a dot blot of immobilizedGLPS F3 contacted with Dectin-1.Fc fusion protein followed by incubationwith goat HRP-conjugated anti IgG1 antibody in the presence varyingamounts of competitor polysaccharides (β-glucan, D-glucose, andD-galactose);

FIG. 3 shows a semi-quantitative analysis of dot blots ofmembrane-immobilized GLPS F3 and GLPS F3C contacted with 27 differentfusion proteins, each comprising the extracellular domain of the listedinnate immunity receptor coupled to IgG1 Fc;

FIG. 4A shows a dot blot of membrane-immobilized GLPS F3 and GLPS F3Cprobed with the 27 fusion proteins listed in FIG. 3. FIG. 4B shows theeffect of EDTA on the binding of Dectin-1.Fc, DC-SIGNR.Fc, KCR.Fc, andTLT-2.Fc to membrane immobilized GLPS F3. FIG. 4C shows a dot blot ofmembrane-immobilized β-glucan probed with Dectin-1.Fc, DC-SIGNR.Fc,KCR.Fc, and TLT-2.Fc fusion proteins;

FIG. 5A shows dot blots of polysaccharide samples probed withDectin-1.Fc, DC-SIGNR.Fc, KCR.Fc, and TLT-2.Fc fusion proteins. FIG. 5Bshows the identity of the sample numbers and provides the dot densitiesof FIG. 5A in semi-quantitative form;

FIG. 6A shows the amount of biotinylated GLPS-F3 coated onto amicrotiter plate as measured using a peroxidase-conjugated avidin assayand reading at OD450 nm to detect the yellow-colored reaction product.FIG. 6B depicts in graphical form the affinity of various receptor.Fcfusion proteins for GLPS-F3 immobilized on a microtiter plate. Theabsolute binding of each receptor.Fc fusion protein is depicted on theleft Y axis (as an OD450 nm reading) and the right Y axis depicts therelative binding in comparison to the binding of Dectin-1.Fc;

FIG. 7 illustrates graphically the percentage binding of variousreceptor.Fc fusion proteins to GLPS-F3 in a competition assay with thepolysaccharides mannan and β-glucan, and with the monosaccharidesD-mannose (Man), D-glucose (Glc), N-acetyl-glucosamine (GlcNAc),D-galactose (Gal), N-acetyl-galactosamine (GalNAc), L-fucose (Fuc) andsialic acid;

FIG. 8A shows graphically the binding of receptor.Fc fusion proteins toDengue Virus, in comparison to a human IgG negative control. FIG. 8Bshows a Western blot of immunocomplexes of Dengue Virus with threereceptor.Fc fusion proteins and a human IgG negative control, probedwith an antibody against the Dengue Virus E protein. FIG. 8C showsgraphically that EDTA inhibits the binding of Dengue Virus to DC-SIGN.Fcfusion protein, but not the binding to DVLR1/CLEC5A.Fc fusion protein.FIG. 8D shows the binding of a DVLR1/CLEC5A.Fc fusion protein to DengueVirus treated with PNGaseF, dithiothreitol (DTT), heat, or UVirradiation, and to untreated Dengue Virus (non);

FIG. 9A shows the expression of DVLR1/CLEC5A in various immune celltypes by flow cytometry using an anti-DVLR1/CLEC5A antibody. Expressionof DVLR1/CLEC5A is indicated where the DVLR1/CLEC5A profile (dotted linetrace) does not match the antibody isotype control (shaded area).

FIG. 9B shows the expression of DC-SIGN in various immune cell types byflow cytometry using an anti-DC-SIGN antibody. Expression of DC-SIGN isindicated where the DC-SIGN profile (dotted line trace) does not matchthe antibody isotype control (shaded area);

FIG. 10A shows flow cytometry analysis of the expression of NS3 proteinusing an anti-NS3 antibody in CD14+ macrophages contacted with live orUV irradiated (UV-DV) Dengue Virus, in comparison to a matched antibodyisotype control (shaded area). FIG. 10B shows graphically extracellularDengue virus titers over time for CD14+ macrophages infected with DengueVirus at different multiplicities of infection (MOI) or with UVirradiated Dengue Virus. FIG. 10C shows an immunoblot illustrating totalDAP12 and phosphorylated DAP12 in CD14+ macrophages infected with Denguevirus at different MOIs. FIG. 10D shows an immunoblot illustrating totalDAP12 and phosphorylated DAP12 in CD14+ macrophages infected with Denguevirus at different times following infection with live Dengue virus orUV irradiated Dengue virus at MOI=5;

FIG. 11 shows an immunoblot illustrating total DAP12 and phosphorylatedDAP12 in CD14+ macrophages electroporated with pLL3.7 vector (control)or with DVLR1/CLEC5A-shRNA prior to infection with Dengue virus;

FIG. 12A shows the secretion of TNF-α at 6 hours after infection ofCD14+ macrophages with live or UV-irradiated Dengue Virus at thespecified MOIs. FIG. 12B shows the secretion of TNF-α at 12 hours afterinfection of CD14+ macrophages with live or UV-irradiated Dengue Virusat the specified MOIs. FIG. 12C shows time course measurements of thesecretion of TNF-α following infection of CD14+ macrophages;

FIG. 13A shows the expression of DC-SIGN and DVLR1/CLEC5A by Westernblot in CD14+ macrophages transfected with DC-SIGN-shRNA orDVLR1/CLEC5A-shRNA, or with vector controls (pWTSI and pLL3.7). FIG. 13Bshows flow cytometry analysis of NS3 expression (using anti-NS3antibody) in CD14+ macrophages electroporated with DC-SIGN-shRNA,DVLR1/CLEC5A-shRNA, or pLL3.7 vector control prior to infection withDengue virus. The shaded area is an isotype control for the NS3antibody. FIG. 13C illustrates a time course analysis of virus titer inthe supernatant of CD14+ macrophages electroporated with DC-SIGN-shRNA,DVLR1/CLEC5A-shRNA, or vector controls, prior to infection with Denguevirus at t=0;

FIG. 14A shows a time course analysis of the secretion of variouscytokines by CD14+ macrophages that were electroporated withDC-SIGN-shRNA, DVLR1/CLEC5A-shRNA, or vector controls prior to infectionwith Dengue virus at t=0. FIG. 14B shows a time course analysis for thecytokine IFN-α under the same conditions;

FIG. 15 shows ELISA measurements of TNF-α secreted into culturesupernatants by CD14+ macrophages infected with Dengue virus and treatedwith the specified monoclonal antibody against DVLR1/CLEC5A at thespecified concentrations;

FIG. 16 a illustrates graphically the binding of various receptor.Fcfusion proteins to Dengue virus. FIG. 16 b shows a Western blot ofimmunocomplexes of Dengue virus with three receptor.Fc fusion proteinsand a human IgG negative control, probed with an antibody against theDengue Virus E protein. FIG. 16 c shows graphically that EDTA inhibitsthe binding of Dengue Virus to DC-SIGN.Fc fusion protein, but not thebinding to DLVR1/CLEC5A fusion protein. FIG. 16 d shows the increase inDengue Virus binding to human 293T cells with the addition of DC-SIGN.Fcand DLVR1/CLEC5A fusion proteins.

FIG. 16 e shows graphically that the addition of various sugars inhibitsthe binding of Dengue Virus to DC-SIGN.Fc fusion protein. FIG. 16 fillustrates graphically the effect of PNGaseF on Dengue Virus binding toDLVR1/CLEC5A fusion protein;

FIG. 17 a illustrates the expression pattern of DC-SIGN in human PBMCs.FIG. 17 b illustrates the expression pattern of DLVR1/CLEC5A in humanPBMCs;

FIG. 18 a shows an immunoblot illustrating dengue virus-induced DAP12phosphorylation (2 h p.i.) in human macrophages using antibodies tophosphotyrosine and DAP12. FIG. 18 b shows immunoblots illustrating thekinetics of DAP12 phosphorylation induced by Dengue Virus andUV-inactivated Dengue Virus. FIG. 18 c shows immunoblots illustratingthe ability of shRNAs to knock down the expression of DLVR1/CLEC5A andDC-SIGN.Fc fusion proteins and to inhibit Dengue virus(m.o.i.=5)-mediated DAP12 phosphorylation. FIG. 18 d shows the effectsof shRNAs on Dengue Virus entry and replication in macrophages. FIG. 18e shows the effect of anti-DLVR1/CLEC5A mAb, anti-DCSIGN mAb and mouseIgG on the expression of nonstructural protein NS3. FIG. 18 fillustrates graphically a time course analysis of the effect of shRNAson the Dengue Virus titers of infected macrophages;

FIG. 19 a illustrates graphically the dose-dependency of Dengue Virusand UV-inactivated Dengue Virus induced TNF-α secretion by machorphages.FIG. 18 b illustrates graphically the kinetics of TNF-α expression afterDengue Virus infection (m.o.i.=5). FIG. 19 c illustrates graphically theeffects of DLVR1/CLEC5A and DC-SIGN shRNAs on the secretion of TNF-α,IL-6, MIP1-α, IL-8, IP-10, and IFN-α from Dengue Virus-infectedmacrophages (m.o.i.=5). FIG. 19 d illustrates graphically the effects ofknock-down experiments using specific shRNAs on the various secretionpathways for Dengue Virus-induced TNF-α and IFN-α secretion. FIG. 19 eillustrates graphically the inhibition of TNF-α secretion in response toDengue Virus serotypes 1-4 by antagonistic anti-DLVR1/CLEC5A mAbs. M.R.mAb (anti-1-mannose receptor mAb, mIgG1) and murine IgM (mIgM) were usedas negative controls;

FIG. 20 a illustrates the expression of NS3 of macrophages infected withdengue virus, dengue virus/anti-E, and dengue virus/anti-prMimmunocomplexes. FIG. 20 b illustrates the level of TNF-α and IFN-αsecretion of macrophages infected with DV2. FIG. 20 c illustrates thelevel of TNF-α and IFN-α secretion of macrophages infected withanti-prM/dengue virus and anti-E/dengue virus immunocomplexes;

FIG. 21 a illustrates graphically time course analysis of thepermeability of HMEC-1 monolayers and TNF-α levels in the supernatants.

FIG. 21 b illustrates graphically the inhibitory effects of TNFR2.Fc andanti-DLVR1/CLEC5A on permeabilisation of endothelial monolayers;

FIG. 22 a illustrates graphically the binding affinity of dengue viruswith human and murine DLVR1/CLEC5A.Fc fusion protein. FIG. 22 b showsthe expression of mDLVR1/CLEC5A in murine splenocytes. FIG. 22 c showsthe expression of mDLVR1/CLEC5A in murine bone marrow (BM)-derivedmacrophage and the murine macrophage cell line Raw264.7;

FIG. 23 a illustrates graphically a comparison of TNF-α release frombinding of dengue virus to murine macrophage cell line Raw264.7 andRaw264.7 cells stably expressing human DC-SIGN. FIG. 24 b illustratesgraphically the secretion of TNF-α by Raw 264.7 cells stably expressinghuman DC-SIGN incubated with DV2 in the presence of mAbs. FIG. 24 cillustrates graphically the inhibition of DV2-induced TNF-α release in adose-dependent manner by anti-mDLVR1/CLEC5A mAbs (3D2H6 and 10D7H3);

FIG. 24 illustrates Kaplan-Meier survival curves of STAT^(−/−) micechallenged with DV2/PL046 and DV2/NGC-C strains;

FIG. 25 a illustrates the effect of mAb 3D2H6 and 10D7H3, raised againstmurine DLVR1/CLEC5A, towards subcutaneous and intestinal hemorrhaging ofDengue Virus-challenged STAT1^(−/−) mice. FIG. 20 b illustrates theeffect of mAbs against DLVR1/CLEC5A (3D2H6 and 10D7H3) towards plasmaleakage into the vital organs of Dengue Virus-challenged STAT₁ ^(−/−)mice.

FIG. 20 c illustrates graphically vascular permeability of vital organsby extraction of Evan blue from organs. FIG. 20 d illustrates the serumlevels of TNF-α and IP-10 and virus titers for Dengue Virus-challengedSTAT1^(−/−) mice in the presence or absence of anti-DLVR1/CLEC5A mAbs orTNFR2.Fc. FIG. 20 e illustrates graphically the survival ofSTAT1-deficient mice challenged with DV2 in the presence of antagonisticanti-murine DLVR1/CLEC5A mAbs or TNFR2.Fc; and

FIG. 26 illustrates that DLVR1/CLEC5A is involved in JEV-mediated DAP12phosphorylation and TNF-α secretion from human macrophages. In FIG. 26a, interaction of DLVR1/CLEC5A.Fc (1 μg) with JEV and dengue virus (DV)(5×10⁶ PFU), respectively, were determined by ELISA. DV interacts withhuman DLVR1/CLEC5A (188 amino acid in length), but not the alternativelyspliced form sDLVR1/CLEC5A (aa 43-65 is deleted). In contrast, JEV onlyinteracts with sDLVR1/CLEC5A, but not full length DLVR1/CLEC5A. In FIG.26 b, dengue virus induces DAP12 phosphorylation (at 2 h p.i.) in humanmacrophages. DAP12 in DV-infected macrophages were precipitated byanti-DAP12 mAb, blotted to nitrocellulose paper after fractionation onSDS-PAGE, followed by incubation with antibodies against phosphotyrosineand DAP12, respectively. JEV-induced DAP12 phosphorylation (m.o.i.=5) isinhibited by pLL3.7/DLVR1/CLEC5A. In FIG. 26 c, kinetics of TNF-αsecretion are shown from human macrophages in response to JEV infection(left). JEV-induced TNF-a secretion is inhibited by pLL3.5/DLVR1/CLEC5AmAb (right). Data are expressed as the mean±s.d. of three independentexperiments.

DETAILED DESCRIPTION

In the following detailed description of embodiments of the invention,reference is made to the accompanying drawings in which like referencesindicate similar elements, and in which is shown by way of illustrationspecific embodiments in which the invention may be practiced. Theseembodiments are described in sufficient detail to enable those skilledin the art to practice the invention, and it is to be understood thatother embodiments may be utilized and that logical, mechanical,biological, electrical, functional, and other changes may be madewithout departing from the scope of the present invention. The followingdetailed description is, therefore, not to be taken in a limiting sense,and the scope of the present invention is defined only by the appendedclaims. As used in the present disclosure, the term “or” shall beunderstood to be defined as a logical disjunction and shall not indicatean exclusive disjunction unless expressly indicated as such or notatedas “xor.”

In one implementation, the disclosure provides fusion proteinscomprising a carbohydrate recognition domain of an innate immunityreceptor and a heterologous polypeptide. By innate immunity receptor ismeant:

1) receptors encoded by genes within the leukocyte receptor complex(LRC) and LRC-related genes on human chromosome 19, including, but notlimited to, the CD66 family (CEACAM1 and PSG1), the SIGLEC family, NGK7,FCGRT, the ILT/LILRA/LILRB (CD85) family, the LAIR family, the KIR(CD158) family (including the KIR2DL subfamily, KIR2DS subfamily, andKIR3DL subfamily), FCAR (CD89), NKp46 (NCR1), and GPVI (GP6); and

2) receptors encoded by genes within the natural killer receptor complex(NKC) on human chromosome 12, including but not limited to MAFA-L(KLRG1), A2M, NKR-PiA (KLRB1), LLt1 (CLEC2D), CD69 (CLEC2C), KLRF1, AICL(CLEC2B), CLEC-2 (CLECFS2), Lox-1 (OLR1), CD94 (KLRD1), NKG2-D (KLRK1),NKG2-F (KLRC4), NKG2-E (KLRC3), NKG2-C (KLRC2), NKG2-A (KLRC1), Ly49L(KLRA1) and PRB3; and

3) all human and mouse C-type lectin (CLEC) family genes, all humanSialic Acid Binding Ig-Like (SIGLEC) genes, all human TriggeringReceptor Expressed on Myeloid Cells (TREM) genes, all human TREM-like(TREML/TLT) genes, all human Toll-Like Receptor (TLR) genes, and allhuman Fc Receptor-like (including FCRL1 through FCLR6, and also FCLRM1and FCLRM2) genes found on human chromosomes.

Additional genes within these groupings that may be used in the methodsof the disclosure may be found using the Human Genome Organization(HUGO) search engine website. See also the locus descriptions inImmunological Reviews 2001 Vol. 181: 20-38, incorporated herein byreference in its entirety.

Orthologues of any of the aforementioned genes from non-human speciesmay be also be used in the methods of the disclosure.

C-type lectin genes that are contemplated for use in the presentdisclosure include, but are not limited to the following human genes,:ASGR1, ASGR2 (CLEC4H2), CD207 (CLEC4K/Langerin), CD209 (DC-SIGN/CLEC4L),CD302 (CLEC13A), CLEC1A, CLEC1B (CLEC-2), CLEC2A, CLEC2B, CD69, CLEC2D,CLEC2L, CLEC3A, CLEC3B, CLEC3O, CLEC3Q, CLEC4A, CLEC4C, CLEC4D (CLEC-6),CLEC4E, CLEC4F (KCLR), CLEC4G, CLEC4M (DC-SIGNR), CD209, DLVR1/CLEC5A,CLEC6A (Dectin-2), CLEC7A (Dectin-1), CLEC9A, CLEC10A, CLEC11A, CLEC12A,CLEC14A, FCER2, KLRB1, KLRF1, LY75 (DEC205), MRC1, MRCIL1, MRC2(Endol80), OLR1, PLA2R1, DCAL1, and COLEC10. Homologues of any of thesegenes are also contemplated, as are orthologues from other animalspecies such as mice and rats. Homologues and orthologues may be 50%,70%, 80%, 80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to any of the enumerated C-type lectin genes. A specificallycontemplated orthologue is the Kupffer Cell Receptor (mKCR) gene in mice(homologous to human CLEC4F).

TREM genes and TREML genes that are contemplated for use in the presentdisclosure include, but are not limited to the following human genes:CD300 Antigen Like Family Member B (CD300 LB), CD300 Antigen Like FamilyMember G (CD300LG), TREM1, TREM2, TREML1 (TLT1), TREML2 (TLT2), TREML3(TLT3), and TREML4 (TLT4). Homologues of any of these genes are alsocontemplated, as are orthologues from other animal species such as miceand rats. Homologues and orthologues may be 50%, 70%, 80%, 80.6%, 83%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any ofthese enumerated genes. Specifically contemplated orthologues includemTREM1, mTREM2, mTLT1, and mTLT4 from mouse.

TLR genes that are contemplated for use in the present disclosureinclude, but are not limited to, the following human genes: TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, andTLR13. Homologues of any of these genes are also contemplated, as areorthologues from other animal species such as mice and rats. Homologuesand orthologues may be 50%, 70%, 80%, 80.6%, 83%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, or 99.9% identical to any of the enumerated TLRgenes.

SIGLEC genes that are contemplated for use in the present disclosureinclude, but are not limited to, the following human genes: CD22, CD33,Myelin Associated Glycoprotein (MAG), SIGLEC5, SIGLEC6, SIGLEC7,SIGLEC8, SIGLEC9, SIGLEClo, SIGLEC11, SIGLEC12, SIGLEC13, andSialoadhesin (SN). Homologues of any of these genes are alsocontemplated, as are orthologues from other animal species such as miceand rats. Homologues and orthologues may be 50%, 70%, 80%, 80.6%, 83%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any ofthe enumerated SIGLEC genes.

Other innate immunity receptors suitable for use in the instantdisclosure include those recited in the Examples below.

The fusion protein may comprise the entire extracellular domain of theinnate immunity receptor, including a carbohydrate recognition domain,or it may comprise a portion of the extracellular domain, including acarbohydrate recognition domain, or it may comprise only a carbohydraterecognition domain.

The heterologous polypeptide may comprise any polypeptide to which acarbohydrate recognition domain of an innate immunity receptor may befused such that the heterologous polypeptide does not interfere with thebinding of a carbohydrate domain to its cognate specific carbohydrate,either in vivo or in vitro. Preferably, the heterologous polypeptide isan immunoglobulin, such as human IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM,IgE, IgD, IgAa, and IgA2, or an immunoglobulin from other animalspecies. Preferably, a fragment of an immunoglobulin is used as theheterologous polypeptide, for example an Fc fragment of an IgG. Inpreferred embodiments, the heterologous polypeptide is an immunoglobulinvariant that does not bind to human Fc receptors. Such variants are wellknown in the art. For example, a human IgG1 Fc variant comprising thefollowing mutations may be used: L234A, L235E, G237A, and P331S.

The heterologous polypeptides may further comprise one or morefunctional domains that permit the fusion polypeptide to be immobilizedon a solid support, or purified from a complex mixture. By way ofexample, the heterologous polypeptide may comprise a His6 tag to permitattachment of the fusion protein to a Ni-NTA solid support according tomethods well known in the art. Also by way of example, the heterologouspolypeptide may comprise a glutathione-5-transferase domain so that theresulting fusion protein can be adsorbed onto, for example, glutathionebeads or glutathione derivatized microtiter plates.

The heterologous polypeptide may also comprise one or more biotins, orbiotin derivatives. In this way, fusion proteins may be immobilized tostreptavidin-conjugated solid supports, or a streptavidin-conjugatedenzyme may be bound to the fusion protein.

The fusion protein may optionally further comprise a linker between theheterologous polypeptide and a carbohydrate recognition domain of theinnate immunity receptor. The linker may be a peptide linker, or it maybe a non-peptidic linker, such as a polyethylene glycol.

The carbohydrate recognition domain may be C-terminal relative to theheterologous polypeptide or it may be N-terminal relative to theheterologous polypeptide in the fusion protein.

The fusion proteins of the disclosure may be prepared by any methodknown in the art for the production of proteins. Preferably, the fusionproteins are prepared using recombinant DNA technology and proteinexpression technology well known in the art. For example, DNA encodingthe carbohydrate recognition domain of an innate immunity receptor maybe obtained by reverse-transcriptase PCR (RT-PCR) of mRNA using primersspecific for the carbohydrate recognition domain of the particularinnate immunity receptor of interest. The resulting DNA may then becloned into an expression vector in frame with DNA encoding theheterologous polypeptide sequence. Expression vectors useful in thepresent disclosure typically contain an origin of replication, apromoter located 5′ to (i.e., upstream of) and followed by the DNAsequence coding for the fusion protein, transcription terminationsequence, and the remaining vector. The expression vectors may alsoinclude other DNA sequence known in the art, for example, stabilityleader sequences that provide for stability of the expression product,secretory leader sequences which provide for secretion of the expressionproduct, and sequences which allow expression of the fusion protein tobe modulated or induced. The expression vector may also contain viralsequences that allow the fusion protein to be expressed using a viralexpression system, such as the baculovirus expression system well knownin the art. The expression vector may be introduced into host cells,such as microbial cells, yeast cells, mammalian cells, or insect cells.The expression vector may be introduced into cells as naked DNA, or itmay be encapsulated within a virus (such as a baculovirus). Theexpression vector may be maintained within the host cell, or it mayintegrate into the host cell genome.

Preferably, the expression vector comprises DNA sequence that lead tothe addition of a secretory leader sequence on the fusion protein,thereby causing the fusion protein to be secreted into the mediumsurrounding the host cells. The fusion protein can then be purified fromthe medium using techniques known in the art. By way of example, if thefusion protein comprises IgG as the heterologous polypeptide, then aProtein A column may be used to bind to the fusion protein to permit thefusion protein to be partitioned from other proteins in the surroundingmedium.

Fusion proteins may also be produced by in vitro translation of a mRNAencoding the fusion protein using an in vitro expression system, such asa Xenopus oocyte expression system.

In an embodiment, the fusion proteins are produced separately and thencoupled to one another using chemical techniques known in the art. Forexample, the carbohydrate recognition domain and the heterologouspolypeptide may be produced separately and then coupled to one anotherusing glutaraldehyde.

Following production of the fusion protein, the fusion protein may belabeled with a detectable label, such as a fluorophore, radiolabel, anenzyme, an enzyme substrate, a dye, a chemiluminescent agent, a magneticparticle, a quantum dot, or any other moiety that produces, directly orindirectly, a detectable signal. Many methods for the conjugation ofsuch detectable labels to proteins are known in the art. By way ofexample only, an N-hydroxysuccinimide-activated dye, most preferably anN-hydroxysuccinimide-activated fluorophore, may be conjugated to thefusion protein by reaction with primary amines on the fusion protein.

In some embodiments, the fusion protein is biotinylated using methodsknown in the art such that the fusion protein comprises one or morebiotins, or one or more biotin derivatives. In this way, the fusionprotein may be attached to a streptavidin-detectable moiety conjugate,such as an enzyme-streptavidin conjugate.

In one series of embodiments, the fusion proteins of the disclosure areused to determine whether a specific carbohydrate component is presentin a composition that comprises a polysaccharide. The methods involvecontacting the polysaccharide with a fusion protein that binds to aspecific carbohydrate component of a polysaccharide, and thendetermining whether the fusion protein has bound to polysaccharide inthe composition. For example, it is known that the carbohydraterecognition domain of CLEC7A (also known as Dectin-1), can interact withβ-1,3-D-glycans (see Brown, G. D. and Gordon, S., 2001, Nature 413,36-7, incorporated herein by reference in its entirety). Binding of afusion protein comprising the carbohydrate recognition domain of CLEC7Ato a polysaccharide composition therefore indicates that thepolysaccharide composition comprises β-1,3 glucan. Similarly, since therodent Kupffer cell receptor (KCR; homologous to human CLEC4F) has highaffinity to D-galactose and N-acetylgalactosamine, and is able to clearD-galactose and D-fucose terminated glycoproteins from serum (seeFadden, A. J., Holt, O. J. and Drickamer, K. (2003), Glycobiology 13,529-37, incorporated herein by reference in its entirety), binding of afusion protein comprising the carbohydrate recognition domain of KCR toa polysaccharide composition therefore indicates that the polysaccharidecomposition comprises D-galactose or N-acetylgalactosamine orD-galactose terminated glycoproteins or D-fucose terminatedglycoproteins. In addition, CD209 (also known as DC-SIGN and CLEC4L) andCLEC4M (also known as DC-SIGNR and L-SIGN) can both bind to Man₉ GlcNAc₂Asn glycopeptide, but only CD209 and not CLEC4M can bind to glycans witha terminal fucose residue (see Guo et al (2004) Nat Struct Mol Biol 11,591-8); therefore, fusion proteins of CD209 and CLEC4M can discriminatebetween polysaccharide compositions comprising these carbohydratecomponents. The methods and reagents of the disclosure may therefore beused to determine the identity of the carbohydrate components of apolysaccharide composition and to determine the relative amounts ofthose carbohydrate components e.g., to “fingerprint” a polysaccharidecomposition. For example, the methods and reagents of the disclosure maybe used to determine the carbohydrate components of a polysaccharidecomposition that has immunomodulatory activity.

In addition, if one knows the identity of the cells that express theinnate immunity receptors from which the carbohydrate recognition domainof the fusion protein is derived, then the assays disclosed hereinreveal the identity of the cells in the body that bind to thepolysaccharide under investigation. Such knowledge, for example, canhelp reveal the mechanism by which a particular polysaccharidecomposition (such as polysaccharides isolated from Ganoderma lucidum)exerts beneficial or deleterious effects on an organism which comes intocontact with the polysaccharide. It is not necessary to know theidentity of the carbohydrate component bound by the carbohydraterecognition domain in this embodiment.

The binding of the fusion proteins of the disclosure to their cognatecarbohydrate component can be performed by immobilizing the compositioncomprising the polysaccharide to a solid support, and then contactingthe solid support with a fusion protein. Binding of the fusion proteinmay be detected by detecting the presence of the fusion protein on thesurface of the solid support, for example, by detecting the presence ofthe heterologous polypeptide on the surface of the solid support or bydetecting the presence of the carbohydrate recognition domain on thesurface of the solid support. For example, if the heterologouspolypeptide is conjugated to a fluorophore, then the presence of thefluorophore, following washing, on the surface of the solid support isindicative of the presence of the fusion protein, which in turn isindicative of the presence of a polysaccharide comprising the specificcarbohydrate component recognized by the carbohydrate recognition domainof the fusion protein.

As used herein, “solid support” is defined as any surface to whichmolecules may be attached through either covalent or non-covalent bonds.This includes, but is not limited to, membranes (for example,polyvinylidene fluoride (PVDF) membranes), plastics (for example,microtiter plates), paramagnetic beads, charged paper, nylon,Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE,polystyrene, gallium arsenide, gold and silver. Any other material knownin the art that is capable of having functional groups such as amino,carboxyl, thiol or hydroxyl incorporated on its surface, is alsocontemplated. This includes surfaces with any topology, including, butnot limited to, spherical surfaces, grooved surfaces, and cylindricalsurfaces e.g., columns.

The composition comprising a polysaccharide (also referred to herein asa “polysaccharide composition”) can be, without limitation, anycomposition that includes a polysaccharide including, for example, aglycoprotein (including a proteoglycan), a glycolipid, peptidoglycan, amicrobial cell wall, a viral particle, and a fungal cell wall. In otherembodiments, the composition comprising a polysaccharide is apolysaccharide free in solution e.g., a polysaccharide that is notattached to a protein or lipid. As used herein, a “polysaccharide” meansa carbohydrate molecule that comprises two or more monosaccharides.

Immobilization of the composition comprising a polysaccharide on a solidsupport may be achieved, for example, by biotinylating thepolysaccharides in the composition, and then immobilizing on astreptavidin-conjugated solid support. In addition, polysaccharides maybe immobilized on, for example, methanol-activated PVDF membranes. It isspecifically contemplated that the methods of the disclosure can beperformed in a “dot blot” format using dots of polysaccharideimmobilized on a PVDF membrane.

In some embodiments, binding of the fusion protein to an immobilizedpolysaccharide is detected by binding a secondary reagent to the fusionprotein, preferably to the heterologous polypeptide, and then detectingthe presence of the secondary reagent. For example, a biotinylatedfusion protein may be attached to a streptavidin-conjugated enzyme, andthe presence of the enzyme detected by adding a substrate that yields adetectable product. A non-biotinylated fusion protein may be detectedusing, for example, an antibody that binds to the heterologouspolypeptide (such as an anti-IgG antibody if the heterologouspolypeptide is IgG, or a IgG Fc), which secondary antibody is conjugatedto an enzyme. For example, if the enzyme is horseradish peroxidase(HRP), then detection of fusion protein binding may be performed usingthe Enhanced Chemiluminescence (ECL) technique well known in the art.The secondary reagent may also, or alternatively, be conjugated to adetectable label such as a fluorophore or a radionuclide. Many othertechniques are known in the art which may be used to detect the bindingof the disclosed fusion proteins to a solid support.

It is specifically contemplated that the aforementioned assays may becarried out in a multiplexed array format. For example, a solid supportmay be partitioned into a plurality of spatially discrete addresses ontowhich a plurality of different compositions may be bound. Then the solidsupport may be contacted with a fusion protein, and the binding of thefusion protein detected. In this way, it can be determined which, ifany, of the immobilized polysaccharide compositions comprises theparticular carbohydrate component bound by the carbohydrate recognitiondomain of the fusion protein.

In another embodiment, a single composition is immobilized on a solidsupport which is partitioned into a plurality of spatially discreteaddresses. Each address is then contacted with a different fusionprotein, each different fusion protein comprising a differentcarbohydrate recognition domain. Following washing to removenon-specifically bound material, binding of the fusion proteins may thenbe detected as described above; the spatial address of each bindingreaction detected reveals the identity of the fusion protein that hasbound. In this way, the composition can be probed with a number ofdifferent fusion proteins in parallel. In this embodiment, each fusionprotein may comprise the same heterologous polypeptide, thereby allowinga single secondary reagent to simultaneously detect binding at eachaddress. For example, if each fusion protein comprises IgG Fc as theheterologous polypeptide, then either an anti-IgG antibody, or ProteinA, or Protein G, may be used to detect binding of the fusion protein.

The fusion proteins and methods of the disclosure may be used to“fingerprint” any composition which comprises polysaccharides,including, but not limited to, polysaccharide compositions obtained fromherbal preparations, such as polysaccharide-containing fractionsisolated from the fungi Reishi (Ganoderma lucidim), Cordyceps sinensis,and Lentinus edodes; and from the plant Dendrobium huoshanense. Inparticular, it is specifically contemplated that the methods used hereinare used to determine the carbohydrate components of the F3polysaccharide fraction of Reishi polysaccharide (see Wang, et al (2002)Bioorg Med Chem 10, 1057-62; Chen, et al (2004) Bioorg Med Chem 12,5595-601; Chien, et al (2004) Bioorg Med Chem 12, 5603-9.; and Hsu et al(2004) J Immunol 173, 5989-99, each of which is specificallyincorporated herein by reference in its entirety).

The methods provided herein can be used to “fingerprint” complexmixtures that include a number of different polysaccharide compositions,or they can be used on preparations that contain only a singlepolysaccharide species e.g., a single glycoprotein or a singlepolysaccharide.

If one knows the identity of the cells that express the innate immunityreceptor from which the carbohydrate recognition domain is derived, thenthe aforementioned assays reveal which cells in the body bind to thepolysaccharide upon introduction of the polysaccharide composition intothe body. It is then possible to obtain agents that modulate theactivity of the identified innate immunity receptor. For example, agentsthat mimic the structure of the polysaccharide or that potentiate theinteraction of the polysaccharide with the innate immunity receptor maybe generated if interaction of the innate immunity receptor with thepolysaccharide leads to beneficial effects in the body. See the sectionbelow entitled “Modulators.”

In another series of embodiments, the methods and fusion proteins of thedisclosure are used to determine the identity of polysaccharidesdisplayed on the surface of a pathogen, such as a fungal cell, abacterial cell, or a virus, such as an enveloped virus, and alsoincluding but not limited to viruses from the Flaviviridae family.Flaviviridae viruses suitable for use in the methods disclosed hereininclude, but are not limited to, members of the genus Flavivirus (suchas, for example, Dengue virus (DV), West Nile Virus (WNV), Japaneseencephamyelitis virus (JEV), yellow fever virus (YFV), and tick-borneencephamyelitis virus) and members of the genus Hepacivirus (such as,for example, Hepatitis C virus). In one such embodiment, a fusionprotein is immobilized on a solid support (for example, using a ProteinA derivatized solid support if the heterologous polypeptide is IgG or afragment thereof), and the solid support is this contacted with acomposition comprising the pathogen of interest. Following washing, thebinding of the pathogen is then detected using, for example, a secondaryreagent that binds specifically to the pathogen in a manner that doesnot compete with the binding of the fusion protein. For example, asecondary antibody that is specific for the pathogen may be used.Binding of the secondary reagent is then detected as described above(for example using HRP-conjugated secondary antibody), or it may bedetected using a tertiary reagent that binds to the secondary reagent(for example, using an anti-IgG antibody conjugated to HRP if thesecondary reagent is an anti-pathogen IgG). If binding of the secondaryreagent is detected, then this reveals that the pathogen comprises apolysaccharide that comprises the specific carbohydrate componentrecognized by the carbohydrate recognition domain of the fusion protein.

Alternatively, the assay may be performed by immobilizing a reagent thatbinds specifically to the pathogen on a solid support. For example, anantibody which binds to the pathogen can be immobilized on a solidsupport, then contacted with a composition comprising the pathogen. Thesolid support is then contacted with the fusion protein(s), and thebinding of the fusion proteins is then detected as described above(preferably, the fusion protein does not compete for pathogen bindingwith the immobilized reagent). For example, if the heterologouspolypeptide of the fusion protein is IgG Fc, then an anti-IgG antibodycan detect binding of the fusion protein to the pathogen; alternatively,if the fusion protein is conjugated to a detectable label, thendetection of the label is used to detect binding.

It is expressly contemplated that the aforementioned pathogen assays canbe carried out in a multiplexed format using, for example, a pluralityof different fusion proteins simultaneously. For example, an antibodythat binds to the pathogen may be immobilized at a plurality of discreteaddresses on a solid support; then the solid support is contacted with acomposition comprising the pathogen; and then each specific address iscontacted with a different fusion protein, each different fusion proteincomprising a different carbohydrate recognition domain. If each fusionprotein comprises the same heterologous polypeptide, then binding of thefusion protein may be detected using a single reagent that binds to theheterologous polypeptide. For example, if the heterologous polypeptideis IgG Fc, then an anti-IgG antibody can be used to detect binding ofthe fusion protein(s). The spatial address of each binding reaction thenreveals the identity of the fusion protein. Alternatively, a multiplexedassay may be carried out using a plurality of different fusion proteinsimmobilized on the solid support at spatially discrete addresses, bycontacting the solid support with the composition comprising thepathogen, followed by contacting the solid support with a secondaryreagent that binds specifically to the pathogen. For example, if thepathogen is Dengue virus, then the secondary reagent may be an antibodyagainst the E envelope protein. As in all the preceding assays, washingmay be used to remove non-specifically bound material from the solidsupport.

Using the methods disclosed herein, it has been discovered that Denguevirus binds to DVLR1/CLEC5A on the surface of CD14+ macrophages. SeeExample 11. It has further been shown that DVLR1/CLEC5A binding toDengue virus results in the activation of DAP12, which in turn leads tothe release of the proinflammatory cytokines TNF-α, MIP-1α, IFN-α, andIL-8 from macrophages. See Example 12. The release of these cytokines isimplicated in the development of Dengue hemorrhagic fever (DHF) andDengue shock syndrome (DSS).

According to embodiments the methods disclosed herein, it has been shownspecifically that DLVR1/CLEC5A interacts with the dengue virus. SeeExample 16-18. Moreover, it is shown that DLVR1/CLEC5A modulates DAP12phosphorylation, which is believed to modulate, at least in part,release of pro-inflammatory cytokines such as TNF-α. See Example 18.When DLVR1/CLEC5A expression is knocked down in dengue virus infectedcells, phosphorylation of DAP12 is reduced and pro-inflammatory cytokinesecretion, including TNF-α, is reduced without affecting secretion ofviral clearance cytokines such as interferon-α. See Examples 18-19.According to embodiments, knock down of DLVR1/CLEC5A may be accomplishedusing convention RNA-interference techniques, including use of bothsi-RNA and sh-RNA. See Example 18-19.

Knowledge of the identity of the innate immunity receptor(s) thatinteract with a pathogen may then be used to develop agents thatmodulate the activity of the innate immunity receptor. For example,modulators that activate an identified innate immunity receptors may beobtained in order to augment the immune response to a particularpathogen. In cases where interaction of an innate immunity receptor to aparticular polysaccharide composition is detrimental to the body (forexample, when a pathogen causes excessive inflammation), modulators maybe obtained that reduce the activity of the innate immunity receptors.For example, agents (such as antibodies) that block the binding of apathogen to an innate immunity receptor may be used to prevent theoccurrence of an undesirable proinflammatory reaction to infection withsaid pathogen. Similarly, if the screening methods disclosed hereinreveal that a particular pathogen (such as a virus) uses an innateimmunity receptor to gain entry into a cell, then an agent that blocksthe binding of the pathogen to the innate immunity receptor will prevententry of the pathogen into the cell.

According to embodiments of the methods disclosed herein, administrationof interruption agents that reduce available DLVR1/CLEC5A binding sitesis shown to increase survival rates of dengue virus infected mice.According to embodiments, administration of DLVR1/CLEC5A antibodies thatinterfere with binding of DLVR1/CLEC5A to DLVR1/CLEC5A ligands was shownto increase mouse survival rates. See Example 25.

In another series of embodiments, the fusion proteins of the disclosureare used to disrupt or prevent the interaction between a polysaccharideand an innate immunity receptor on a cell surface. In this series ofembodiments, the fusion protein comprises the carbohydrate recognitiondomain of the innate immunity receptor that is expressed on the cellsurface. The cell expressing the innate immunity receptor is thencontacted with the fusion protein, either in vivo or in vitro, wherebythe fusion protein competes with the polysaccharide for binding to theinnate immunity receptor.

If interaction of the polysaccharide with the innate immunity receptoron the cell surface leads to deleterious effects in an organism, then atherapeutically effective amount of the fusion protein may beadministered to the organism in a pharmaceutical composition to preventor diminish the interaction. Preferably, the heterologous polypeptide ofthe administered fusion protein does not bind to any cell surfacereceptor. For example, the heterologous polypeptide may be comprised ofa mutated variant of IgG Fc that does not bind to Fc receptors on cellsurfaces.

Purification

In another series of embodiments, the fusion proteins are used to atleast partially purify or isolate polysaccharides that comprise thespecific carbohydrate component recognized by the carbohydraterecognition domain of the fusion protein. For example, the fusionprotein may be immobilized on a solid support, and a compositionsuspected of containing, or known to contain, a polysaccharidecomposition is contacted with the solid support. If the compositioncomprises a polysaccharide that can bind to the carbohydrate recognitiondomain of the fusion protein, then that polysaccharide will bind to thefusion protein. The solid support can then be washed to removenon-specifically bound components of the composition, and the boundpolysaccharide may then be eluted by dissociating the interaction withthe fusion protein, and collected. For example, if the fusion proteincomprises the carbohydrate recognition domain of a lectin receptor, thenthe interaction may be dissociated using EDTA to chelate Ca²⁺. In thisway, it is possible to purify specific polysaccharide compositions fromcomplex mixtures. In preferred embodiments, this method is used topurify polysaccharides isolated from Ganoderma lucidum (Reishi).

For the aforementioned purification method, the solid support maycomprise, for example, a column to which the fusion protein is bound.Suitable columns include Sepharose Protein A columns, to which fusionproteins comprising IgG as the heterologous polypeptide may be bound viainteraction with of the IgG domain of the fusion protein with Protein A.Alternatively, CNBr activated column media may be bound to fusionproteins.

The present disclosure also provides kits that can be used in any of theabove methods. In one embodiment, a kit comprises a fusion proteinaccording to the disclosure, in one or more containers. The kit may alsocomprise a secondary reagent, such as an antibody that specificallybinds to the heterologous polypeptide domain of the fusion protein e.g.,an anti-IgG antibody if the heterologous polypeptide is IgG, or afragment thereof. The kit may also comprise reagents and buffers fordetecting the binding of a fusion protein to a polysaccharide. Forexample, in embodiments where a HRP-conjugated secondary antibody isused to detect the binding of a fusion protein to a polysaccharide, thekit may comprise the reagents necessary to establish an enhancedchemiluminescence reaction e.g., one or more containers comprisingluminol, p-coumaric acid, Tris buffer, and hydrogen peroxide. The kitmay also comprise one or more positive control polysaccharides. The kitmay also comprise one or more solid supports for use in theaforementioned methods, for example, one or more PVDF membranes or oneor more multiwell microtiter plates.

Modulators

As described above, the methods of the disclosure identify innateimmunity receptor(s) that interact with a particular polysaccharide.This information then allows one to obtain modulators of the identifiedinnate immunity receptor. A modulator can be an agonist, an antagonist(including competitive and non-competitive antagonists), or an inverseagonist of an innate immunity receptor. A modulator may, withoutlimitation: inhibit the binding of a polysaccharide to an innateimmunity receptor; potentiate the binding of a polysaccharide to aninnate immunity receptor; or function as a mimetic of a polysaccharidethat binds to an innate immunity receptor, thereby activating the innateimmunity receptor even in the absence of the polysaccharide.

Modulators of innate immunity receptors include antibodies. For example,an antagonistic antibody against an innate immunity receptor can preventbinding of a pathogen to the innate immunity receptor. In some cases,such an antibody is a neutralizing antibody as it prevents the entry ofthe pathogen into the cell that expresses the innate immunity receptor.Alternatively, an agonistic antibody may function as a mimetic of apolysaccharide composition that exerts a beneficial effect on a cell. Anantagonistic antibody may also bind to an innate immunity receptor insuch a way as to block the downstream signaling by the receptor uponpathogen binding. Antibodies may be, without limitation, polyclonal,monoclonal, monovalent, bispecific, heteroconjugate, multispecific,human, humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above. The term “antibody,” as used herein,refers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds an antigen. Theimmunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD,IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab)or “monoclonal antibody” (Mab) is meant to include intact molecules, aswell as, antibody fragments (such as, for example, Fab and F(ab′)2fragments) which are capable of specifically binding to protein. Fab andF(ab′)2 fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation of the animal or plant, and may have lessnon-specific tissue binding than an intact antibody (Wahl et al., J.Nucl. Med. 24: 316-325 (1983)). Methods for producing antibody agonistsare described in, for example, PCT publication WO 96/40281; U.S. Pat.No. 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998); Chen et al.,Cancer Res. 58 (16): 3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58 (15): 3209-3214(1998); Yoon et al., J. Immunol. 160 (7): 3170-3179 (1998); Prat et al.,J. Cell. Sci. 111 (Pt2): 237-247 (1998); Pitard et al., J. Immunol.Methods 205 (2): 177-190 (1997); Liautard et al., Cytokine 9 (4):233-241 (1997); Carlson et al., J. Biol. Chem. 272 (17): 11295-11301(1997); Taryman et al., Neuron 14 (4): 755-762 (1995); Muller et al.,Structure 6 (9): 1153-1167 (1998); Bartunek et al., Cytokine 8 (1):14-20 (1996); Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981) (which are all incorporated by reference herein in theirentireties).

The disclosure provides non-limiting specific examples ofanti-DVLR1/CLEC5A monoclonal antibodies that prevent TNF-α release frommacrophages following DV infection. See Example 15. These antibodies canbe used in the pharmaceutical compositions and the methods of treatmentspecified herein, particularly in compositions and methods for thetreatment or prophylaxis of DV infection in humans.

The present disclosure also provides humanized anti-DVLR1/CLEC5Aantibodies that prevent TNF-α release from macrophages following DV orJEV infection. See Examples 20-26. In specific embodiments, thehumanized antibody is selected from the group consisting of humanizedantibodies 9B12, 3E12A2, 3E12C1, 3E12G9, and 8H8F5. These antibodies canbe used in the pharmaceutical compositions and the methods of treatmentspecified herein, particularly in compositions and methods for thetreatment or prophylaxis of DV infection in humans. Specific treatmentsinclude inhibiting DV-induced plasma leakage, as well as subcutaneousand vital organ hemorrhaging. The humanized antibodies can be used asmethods of treatment for DV-induced hemorrhagic shock and sepsis. It isexpressly contemplated that the principles set forth herein with respectto the mitigation to cytokine stimulation by virus is applicable to allviruses that bind to and modulate stimulating receptors of cells.

Moreover, the principles of discovery and treatment of viruses can besimilarly extended to cell entry receptors, as well as the action ofbacterial, fungus, and parasites. The methods of the present disclosurewill enable persons of ordinary skill in the art to determine thebinding profiles of pathogens (i.e., which receptors they bind to),determine the effect that binding the receptor has, and provideinterruption agents, such as antibodies, to interfere with the pathogensability to bind to the target receptor.

According to embodiments, the monoclonal antibodies (mAbs) generated byfusion of murine splenocytes and NS1 myeloid partner cells,anti-DLVR1/CLEC5A mAb can be generated by phage display technology togenerate single chain human anti-human DLVR1/CLEC5A mAbs. The agonisticand antagonistic mAbs can be selected based on the screening methoddisclosed in Examples 19-25.

To decrease antigencity of current murine anti-human DLVR1/CLEC5A mAb,the wild type Fc portion is replaced with human immunoglobulin G1(IgG1), according to embodiments. To further abolish Fc binding to Fcreceptor and prevent complement activation, the mutated Fc fragment ofhuman IgG1 (L234A, L235E, G237A, and P331S) may be used to replace thewild-type Fc to generate the humanized mAbs. To further decrease theantigenecity, the framework region of antibody V domain may be replacedwith a human sequence.

Modulators of innate immunity receptors also include small moleculesidentified by high throughput screening methods. Such high throughputscreening methods typically involve providing a combinatorial chemicalor peptide library containing a large number of potential therapeuticcompounds (e.g., ligand or modulator compounds). Such combinatorialchemical libraries or ligand libraries are then screened in one or moreassays to identify those library members (e.g., particular chemicalspecies or subclasses) that bind to the innate immunity receptor ofinterest. The compounds so identified can serve as conventional leadcompounds, or can themselves be used as potential or actualtherapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g., U.S.Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37: 487-493;and Houghton et al., 1991, Nature, 354: 84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90: 6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114: 6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114: 9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116: 2661),oligocarbamates (Cho et al., 1993, Science, 26i: 1303), or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59: 658), nucleicacid libraries (for example, see U.S. Pat. No. 5,270,163 describing thegeneration of nucleic acid ligands, also known as “aptamers”), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14 (3): 309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

Pharmaceutical Compositions

The instant disclosure also provides pharmaceutical compositions. Insome embodiments, the pharmaceutical compositions comprise the fusionproteins of the disclosure. In other embodiments, the pharmaceuticalcompositions comprise a modulator of an innate immunity receptor (forexample antibodies against an innate immunity receptor such asDVLR1/CLEC5A, including the antibodies exemplified in Example 15). Insuch pharmaceutical compositions, the fusion protein or the innateimmunity receptor modulator form the “active compound.” In someembodiment, the pharmaceutical compositions are administered to asubject in order to treat or prevent diseases or disorders characterizedby the binding of a polysaccharide to an innate immunity receptor on thesurface of a cell in that subject. In other embodiments, thepharmaceutical compositions are administered to a subject to activate aninnate immunity receptor in circumstances where increasing the activityof that receptor is beneficial to the subject. In still otherembodiments, the pharmaceutical compositions are administered to asubject to potentiate the binding of a polysaccharide composition to aninnate immunity receptor.

In addition to active compound, the pharmaceutical compositionspreferably comprise at least one pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions. Apharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Subject as used herein refers to humans and non-human primates (e.g.,guerilla, macaque, marmoset), livestock animals (e.g., sheep, cow,horse, donkey, pig), companion animals (e.g., dog, cat), laboratory testanimals (e.g., mouse, rabbit, rat, guinea pig, hamster), captive wildanimals (e.g., fox, deer) and any other organisms who can benefit fromthe agents of the present disclosure. There is no limitation on the typeof animal that could benefit from the presently described agents. Themost preferred subject of the present disclosure is a human. A subjectregardless of whether it is a human or non-human organism may bereferred to as a patient, individual, animal, host, or recipient.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water-soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions, are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, or adjuvant materials can beincluded as part of the composition. The tablets, pills, capsules,troches and the like can contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in subjects. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the disclosure, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in subjects. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of an activecompound of the disclosure may range from about 0.001 to 30 mg/kg bodyweight, preferably about 0.01 to 25 mg/kg body weight, more preferablyabout 0.1 to 20 mg/kg body weight, and even more preferably about 1 toto mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg bodyweight. Without limitation, the active compound can be administeredbetween one time per week and three or more times per day, for betweenabout 1 to 10 weeks, preferably between 2 to 8 weeks, more preferablybetween about 3 to 7 weeks, and even more preferably for about 4, 5, or6 weeks. The skilled artisan will appreciate that certain factors caninfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a pharmaceutical composition of thedisclosure can include a single treatment or, preferably, can include aseries of treatments.

Gene Therapy and RNAi

Constructs encoding the fusion proteins of the disclosure can be used asa part of a gene therapy protocol to deliver therapeutically effectivedoses of a receptor fusion protein to a subject. A preferred approachfor in vivo introduction of nucleic acid into a cell is by use of aviral vector containing nucleic acid, encoding a fusion protein of thedisclosure. Infection of cells with a viral vector has the advantagethat a large proportion of the targeted cells can receive the nucleicacid. Additionally, molecules encoded within the viral vector, e.g., bya cDNA contained in the viral vector, are expressed efficiently in cellswhich have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous nucleicacid molecules encoding fusion proteins in vivo. These vectors provideefficient delivery of nucleic acids into cells, and the transferrednucleic acids are stably integrated into the chromosomal DNA of thehost. The development of specialized cell lines (termed “packagingcells”) which produce only replication-defective retroviruses hasincreased the utility of retroviruses for gene therapy, and defectiveretroviruses are characterized for use in gene transfer for gene therapypurposes (for a review see Miller, A. D. (1990) Blood 76:27 1). Areplication defective retrovirus can be packaged into virions which canbe used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.,(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals.

Another useful viral gene delivery system uses adenovirus-derivedvectors. The genome of an adenovirus can be manipulated such that itencodes and expresses a gene product of interest but is inactivated interms of its ability to replicate in a normal lytic viral life cycle.See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeldet al., Science 252:431-434 (1991); and Rosenfeld et al., Cell68:143-155 (1992). Suitable adenoviral vectors derived from theadenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g.,Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinantadenoviruses can be advantageous in certain circumstances in that theyare not capable of infecting nondividing cells and can be used to infecta wide variety of cell types, including epithelial cells (Rosenfeld etal., (1992) cited supra). Furthermore, the virus particle is relativelystable and amenable to purification and concentration, and as above, canbe modified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated, into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al., cited supra;Haj-Ahmand et al., J. Virol. 57:267 (1986)).

In another embodiment, non-viral gene delivery systems of the presentdisclosure rely on endocytic pathways for the uptake of the subjectnucleotide molecule by the targeted cell. Exemplary gene deliverysystems of this type include liposomal derived systems, poly-lysineconjugates, and artificial viral envelopes. In a representativeembodiment, a nucleic acid molecule encoding a fusion protein of thedisclosure can be entrapped in liposomes bearing positive charges ontheir surface (e.g., lipofectins) and (optionally) which are tagged withantibodies against cell surface antigens of the target tissue (Mizuno etal. (1992) No Shinkei Geka 20:547-551; PCT publication WO91%6309;Japanese patent application 1047381; and European patent publicationEP-A43075).

Gene delivery systems for a gene encoding a fusion protein of thedisclosure can be introduced into a subject by any of a number ofmethods. For instance, a pharmaceutical preparation of the gene deliverysystem can be introduced systemically, e.g., by intravenous injection,and specific transduction of the protein in the target cellsoCcurspredominantly from specificity of transfection provided by the genedelivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of thereceptor gene, or a combination thereof. In other embodiments, initialdelivery of the recombinant gene is more limited with introduction intothe animal being quite localized. For example, the gene delivery vehiclecan be introduced by catheter (see U.S. Pat. No. 5,328,470) or bystereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3 054-3057).The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Where the fusion protein can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the fusionprotein.

In another embodiment, the expression of an innate immunity receptorthat is identified according to the methods disclosed herein as beinginvolved in the pathogenesis is reduced or completely inhibited usingRNA interference (RNAi). RNAi is well known in the art and may beaccomplished using small interfering RNA (siRNA). siRNAs according tothe invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps,15 bps, 10 bps, 5 bps or any integer thereabout or therebetween. SuchsiRNAs can be administered, e.g., in a form encoded by a vector (forexample, a vector encoding a small hairpin RNA (shRNA)) or as a liposomenucleic acid complex. The preparation of lipid:nucleic acid complexes,including targeted liposomes such as immunolipid complexes, is wellknown to one of skill in the art (see, e.g., Crystal, Science270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995);Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al.,Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722(1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728,4,774,085, 4,837,028, and 4,946,787). Accordingly, the presentdisclosure also provides pharmaceutical compositions comprising RNAmolecules that are capable of mediating RNA interference of an innateimmunity receptor when administered to a subject.

According to embodiments, the present disclosure provides a non-limitingexample of the RNAi-mediated “knock down” of the DVLR1/CLEC5A gene inmacrophages. The attenuation of DVLR1/CLEC5A in this mannersignificantly reduces the secretion of proinflammatory cytokines inDV-infected macrophages, thereby indicating that RNAi-mediatedattenuation of DVLR1/CLEC5A will be useful for the treatment of DV.

It is specifically contemplated that siRNA or shRNA that attenuatesexpression of DVLR1/CLEC5A is used for the RNAi-mediated treatment ofsubjects infected with Dengue virus. Methods for designing,synthesizing, and administering shRNA and siRNA in order to attenuatethe expression of a specific gene are well known in the art and aredescribed in, for example, U.S. Pat. No. 7,022,828. Non-limitingexamples of agents suitable for formulation with the shRNA constructsand siRNA molecules of the disclosure include: PEG conjugated nucleicacids, phospholipid conjugated nucleic acids, nucleic acids containinglipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (suchas Pluronic P85) which can enhance entry of drugs into various tissues,for example the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin.Pharmacol., 13, 16 26); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release deliveryafter implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47 58)Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as thosemade of polybutylcyanoacrylate, which can deliver drugs across the bloodbrain barrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941949, 1999). Othernon-limiting examples of delivery strategies, including CNS delivery ofthe nucleic acid molecules of the instant disclosure include materialdescribed in Boado et al., 1998, J. Pharm. Sci., 87, 1308 1315; Tyler etal, 1999, FEBS Lett., 421, 280 284; Pardridge et al., 1995, PNAS USA.,92, 5592 5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73 107;Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910 4916; andTyler et al., 1999, PNAS USA., 96, 7053 7058. All these references arehereby incorporated herein by reference. In addition, compositionscomprising surface-modified liposomes containing poly (ethylene glycol)lipids (PEG-modified, or long-circulating liposomes or stealthliposomes) may also be used with the nucleic acids of the disclosure.Nucleic acid molecules of the disclosure can also comprise covalentlyattached PEG molecules of various molecular weights. These formulationsoffer a method for increasing the accumulation of drugs in targettissues. This class of drug carriers resists opsonization andelimination by the mononuclear phagocytic system (MPS or RES), therebyenabling longer blood circulation times and enhanced tissue exposure forthe encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601 2627;Ishiwata et al., Chem. Pharm, Bull. 1995, 43, 1005 1011). Such liposomeshave been shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 1995, 267, 1275 1276; Oku et al., 1995, Biochim.Biophys. Acta, 1238, 86 90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,24864 24870; Choi et al., International PCT Publication No. WO 96/10391;Ansell et al., International PCT Publication No. WO 96/10390; Holland etal., International PCT Publication No. WO 96/10392; all of which areincorporated by reference herein). Long-circulating liposomes are alsolikely to protect drugs from nuclease degradation to a greater extentcompared to cationic liposomes, based on their ability to avoidaccumulation in metabolically aggressive MPS tissues such as the liverand spleen.

EXAMPLES

The present disclosure is further described by the followingnon-limiting examples:

Example 1 Preparation of Innate Immunity Receptor:Fc Fusion Protein CellCulture

293F cells (Invitrogen; R790-07) were cultured in serum-free 293FREESTYLE 203 expression medium (Invitrogen, 12338-018) in a 125 mLflask on an orbital shaker (125 rpm) at 37° C. in a CO₂ incubator.

Construction of Receptor.Fc Fusion Genes

The extracellular domains of lectin receptors, TREMs and TLTs werecloned by the reverse-transcriptase polymerase chain reaction (RT-PCR),followed by subcloning into a yT&A vector and then into apcDNA3.1(+)hIgG1.Fc expression vector. The resulting receptor.Fcconstruct encodes recombinant proteins that are fused with a mutatedhuman IgG1 Fc portion, which does not bind to human Fc receptors. Themutations in the IgG1 Fc portion are L234A, L235E, G237A, and P331S. Thesequences of the primers used to RT-PCR amplify the extracellulardomains are (alternatively primers can be selected from the sequenceslisted in Table 2):

CLEC ¹ A/CLEC- ¹ sense primer SEQ ID NO: 15′-GAATCCTTTCAGTACTACCAGCTCTCC-3′ antisense primer SEQ ID NO: 25′-GAATTCTCAGTCACCTTCGCCTAATGT-3′ CLEC ¹ B/CLEC- ² sense primerSEQ ID NO: 3 5′-GGATCCCTGGGGATITGGTCTGTC-3′ antisense primerSEQ ID NO: 4 5′-GAATTCTTAAGGTAGITGGTCCAC-3′ CLEC ² B/AICL sense primerSEQ ID NO: 5 5′-GGATCCTCTCAGAGTTTATGCCCC-3′ antisense primerSEQ ID NO: 6 5′-GGATCCCCCCATTATCTTAGACAT-3′ CLEC ⁴ A/DCIR sense primerSEQ ID NO: 7 5′-GGATCCTTTCAAAAATATTCTCAGCTTCTT-3′ antisense primerSEQ ID NO: 8 5′-GAATTCTCATAAGTGGATCTTCATCATC-3′ CLEC ⁴ C/BDCA- ²sense primer SEQ ID NO: 9 5′-GGATCCTTTATGTATAGCAAAACTGTCAAG-3′antisense primer SEQ ID NO: 10 5′-GAATTCTTATATGTAGATCTTCTTCATCTT-3′ CLEC⁴ D/CLEC- ⁶ sense primer SEQ ID NO: 11 5′-GAATCCCATCACAACTTTTCACGCTGT-3′antisense primer SEQ ID NO: 12 5′-GAATTCCTAGTTCAATGTTGTTCCAGG-3′ CLEC ⁴E/MINCLE sense primer SEQ ID NO: 13 5′-GAAGATCTACATTTCGCATCTTTCAAACC-3′antisense primer SEQ ID NO: 14 5′-GCGGTTAAAGAGATTTTCCTTTGTTCA-3′ CLEC ⁴K/Langerin sense primer SEQ ID NO: 15 5′-GGATCCCGGTTTATGGGCACCATA-3′antisense primer SEQ ID NO: 16 5′-GGATCCTCACGGTTCTGATGGGAC-3′ CLEC ⁴L/DC-SIGN sense primer SEQ ID NO: 17 5′-GGATCCAAGGTCCCCAGCTCCATAAG-3′antisense primer SEQ ID NO: 18 5′-GAATTCCTACGCAGGAGGGGGGT-3′CLEC4M/DC-SIGNR/L-SIGN sense primer SEQ ID NO: 195′-GGATCCTCCAAGGTCCCCAGCTCC-3′ antisense primer SEQ ID NO: 205′-GAATTCCTATTCGTCTCTGAAGCAGG-3′ DLVR ¹ /CLEC5A (MDL- ¹ ) sense primerSEQ ID NO: 21 5′-AGATCTAGTAACGATGGTTTCACCAC-3′ antisense primerSEQ ID NO: 22 5′-GAATTCCTGTGATCATTTGGCATTCTT -3′ CLEC6A/Dectin- ²sense primer SEQ ID NO: 23 5′-GGATCCACATATGGTGAAACTGGC-3′antisense primer SEQ ID NO: 24 5′-GAATTCCATCAGTCGATGGGC-3′ CLEC ⁷A/Dectin-1 sense primer SEQ ID NO: 25 5′-GGATCCACCATGGCTATTTGGAGATCC-3′antisense primer SEQ ID NO: 26 5′-GAATTCTTACATTGAAAACTTCTTCTCACA-3′ CLEC¹⁰ A/ML ² sense primer SEQ ID NO: 27 5′-GGATCCTCCAAATTTCAGAGGGACCTG-3′antisense primer SEQ ID NO: 28 5′-GAATTCTCAGTGACTCTCCTGGCTG-3′ CLEC ¹²A/CLL- ¹ sense primer SEQ ID NO: 29 5′-GGATCCGTAACTTTGAAGATAGAAATGAAA-3′antisense primer SEQ ID NO: 30 5′-GAATCCTCATGCCTCCCTAAAATATGTA-3′ CLEC ¹³ A/BIMLEC sense primer SEQ ID NO: 31 5′-GGATCCTCATGCTCCGGGCCGCG -3′antisense primer SEQ ID NO: 32 5′-GAATTCGCTAGCAATCACCAATGCTGA-3′COLEC12/CL-P1 sense primer SEQ ID NO: 33 5′-AGAGGTGACAGAGGATCCCA-3′antisense primer SEQ ID NO: 34 5′-GAATTCGTGATCCCATCACAGTCC-3′MAFA-L/KLRG-1 sense primer SEQ ID NO: 35 5′-GGATCCTGCCAGGGCTCCAACT-3′antisense primer SEQ ID NO: 36 5′-ATGACAGATCTGAGGGTCA-3′

Expression and Purification of Recombinant Receptor.Fc Fusion Proteins

The receptor.Fc proteins were over-expressed using the FREESTYLE 293Expression System (Invitrogen, Carlsbad, Calif.) and purified on proteinA columns. Briefly, 3×10⁷ 293-F cells were spun down at 1,500 rpm, thenresuspended in 28 ml FREESTYLE 293 expression medium. Then, 40 μL of293FECTIN was mixed with 1 ml OPTI-MEM (Invitrogen, 31985-062) for 5 minat room temperature, then incubated with 30 μg plasmid DNA in 1 mlOPTI-MEM (Invitrogen, 31985-062) for another 20 min, before addition tothe 293-F cells. After 48 h, the supernatant was harvested and therecombinant fusion proteins were purified by protein A columns.

Example 2 Preparation of Polysaccharide Extracts Crude Extracts ofReishi

Crude Reishi extract (prepared via alkaline extraction, neutralizationand ethanol precipitation) was obtained from Pharmanex Co. (CA, USA).Spectrapor® dialysis membrane tubing with molecular weight cut off(MWCO) 6000-8000 dalton, Thermo bio-basic SEC-1000 columns, Tosoh TSKG5000PWx1 SEC columns, and all chemicals and reagents were from Sigma,or Aldrich Co., unless indicated.

Purification of Reishi Extract

Crude Reishi powder (6 g) (obtained from Pharmanex Co.) was dissolved in120 mL of ddH₂O, stirred at boiling water (100° C.) for 2 h, andcentrifuged (1000 rpm) for 1 h to remove insoluble material. Theresulting solution was concentrated at between about 40° C. and about50° C. to give a small volume, and then lyophilized to generate 5 g(83%) powder of dark-brown color (G. lucidum polysaccharides; GLPS).This water soluble residue was stored at −20° C. until furtherpurification.

Standardization-Isolation of the F3 Fraction of Reishi Polysaccharide

G. lucidum polysaccharide fraction 3 (hereinafter referred to as “GLPSF3” and “F3”) was isolated from the dark powder of water soluble residueof Reishi polysaccharide. All chromatography steps were performed at 4°C. in a cold room. The sample (2.1 g) was dissolved in a small volume ofTris buffer (pH 7.0, 0.1 N) containing 0.1 N sodium azide, and purifiedby gel filtration chromatography using a Sephacryl S-500 column (95×2.6cm) with 0.1 N Tris buffer (pH 7.0) as the eluent. The flow rate was setat 0.6 mL/min, and 6.0 mL per tube was collected. After chromatography,each fraction was subjected to the phenol-H₂ SO₄ method to detect thecontent of sugar in each tube. Five fractions were collected (fraction1-5). Fraction 3 (F3) was concentrated at about 40˜50° C. in a rotaryvaporizer to give a small volume which was then dialyzed using a6000-8000 dalton MWCO membrane to remove excessive salt and sodiumazide. Following dialysis, F3 was then lyophilized to give 520 mg ofsolid.

Preparation of Polysaccharides from Cordyceps Sinensis

To purify the polysaccharides from Cordyceps sinensis, samples werechopped into 0.2 cm³ pieces then incubated in deionized boiling water(100° C.) for 60 min, then cooled down to room temperature beforepassing through the 0.2 μm filter, followed by addition of an equalvolume of ethanol to precipitate the polysaccharides. The precipitateswere dried using a lyophilizer and stored at 4° C. Total sugar analysisof the polysaccharides was determined by the Phenol-H₂ SO₄ method, bymeasuring OD at 485 nm, while the purity of the polysaccharides wasdetermined by HPLC using a Thermo Bio-Basic SEC-1000 column with UVdetection at 280 nm and using a R1 detector.

Preparation of Polysaccharides from Dendrobium huoshanense

Air-dried D. huoshanense was crushed and ground to a powder, homogenizedin distilled water, and stirred at 40° C. overnight. The insolublematerial was collected by centrifugation. The supernatant wasconcentrated to a small volume, and then added to 1 volume of ethanol toyield a precipitate (O) and supernatant (N). A TSK G-5000 PW sizeexclusion column was used in high performance liquid chromatography(HPLC) for polysaccharides analysis with standard pullulan fractionshaving defined molecular weights. The molecular weight ofpolysaccharides in N was estimated as between 1.2×10⁵-4.1×10⁵ daltons,and the molecular weight of polysaccharides in O was estimated asbetween 1.0×10⁶-2.2×10⁵ daltons. The total carbohydrate content wasmeasured by the phenol-sulfuric acid method. Polysaccharides in O were83%, and polysaccharides in N were 77%. Both O and N test positive withan iodine reaction (λmax 440 nm, deep blue color) suggesting that thepolysaccharides in these fractions are primarily α-D-glucan.

Preparation of Polysaccharides from Mushroom

Air-dried Lentinus edodes was crushed and ground to a powder,homogenized in distilled water, and stirred at 4° C. overnight. Residueswere removed by centrifugation and supernatant was concentrated to asmall volume, then lyophilized to give crude polysaccharide L. Then,0.25N NaOH solution was added to the water insoluble residue (which wasisolated by centrifugation), and the mixture was then stirred at roomtemperature overnight before adding 2 volume of ethanol to precipitatethe polysaccharides. Distilled water was then added to the precipitatedpolysaccharide, followed by acetic acid to neutralize pH. The resultingsolution was centrifuged and lyophilized to give polysaccharide M. HPLCusing a TSK G-500 PW size exclusion column was then performed in orderto analyze the polysaccharides. The total carbohydrates content wasmeasured by the phenol-sulfuric acid method with L comprising 79%carbohydrates, and M comprising 90% carbohydrates. A comparison withdata of the fractions of polysaccharides from Lentinus edodes suggestedthat the polysaccharides L and M are primarily β-1,3-D-glucan.

Preparation of β-1,3-glucan, D-glucose and D-galactose

To prepare samples for a competition assay, 100 mg of β-1,3-glucan(Fluka, Japan) was suspended in 7.5 ml of water, and 50 L of a 40% (w/w)aqueous solution of sodium hydroxide was added. The mixture was heatedunder reflux for 1.5 hours, and cooled. Then, methanol was added toprecipitate β-1,3-glucan. The β-1,3-glucan precipitate was dissolved inwater, dialyzed with 4 L dd-H₂O four times, and concentrated at reducedpressure to obtain the water-soluble β-1,3-glucan. D-Glucose (Sigma) andD-galactose (Sigma) were dissolved in dd-H₂O (100 mg/ml) and stored at4° C.

Preparation of Biotinyl-F3

Reishi polysaccharides-F3 were labeled with biotin using a “one pot”reaction. Specifically, Reishi polysaccharide-F3 (100 mg) in 0.2 NNaHCO₃/Na₂ CO₃ (10 mL) was reacted withbiotinamidohexanoyl-6-amino-hexanoic acid N-hydroxy-succinimide ester(biotin-XX-NHS) 1.0 mg in DMF (1 mL). The mixture was stirred at roomtemperature for 12 h. After completion of the reaction, the resultingsolution was dialyzed using membrane tubing with a MWCO of 6000-8000dalton (5×500 mL) at 4° C. for 48 h. After dialysis, the biotinyl-F3 waslyophilized to give a brown powder 90 mg (90%). The purification ofbiotinyl-F3 was monitored by HPLC and streptavidin-FITC was used for thebinding assay.

Example 3 Western Blot Analysis of Purified Receptor:Fc Fusion Proteins

The purified receptor.Fc fusion proteins of Example 1 were subjected toelectrophoresis, transferred onto nitrocellulose membrane (Hybond-Cextra, Amersham Pharmacia Biotech) and reacted with (1:3000)peroxidase-conjugated goat anti-human IgG Ab (Jackson, Pa., USA) in TBST(5% non-fat dry milk in Tris-buffered saline with 0.02% Tween 20)buffer. After washing with TBST, blots were then incubated with enhancedchemiluminescence reagents (Amersham Pharmacia Biotech) forvisualization.

Example 4 Immunosorbent Dot Binding Assay

Biotinylated F3 was blotted onto methanol-activated PVDF membranes (2μL/dot) after 5-fold serial dilution, using a Bio-Dot MicrofiltrationApparatus™ (Bio-Rad, CA, USA). After drying in air, the blot wasincubated in TBST, followed by incubation with 100 μLstreptavidin-conjugated horseradish peroxidase (HRP) (1:2000 dilution)(Chemicon, CA, USA). Binding reactions were visualized with enhancedchemiluminescence (ECL) reagents (Amersham. Pharmacia Biotech).

Non-biotinylated polysaccharides were also immobilized ontomethanol-activated PVDF membranes, followed by incubation with 100 μLreceptor.Fc fusion protein (1 μg/ml, in 2 mM CaCl₂/TBST) on a Bio-DotMicrofiltration Apparatus™ (Bio-Rad, CA, USA) for 1 h at roomtemperature, then followed by reaction with (1:3000) HRP-conjugated goatanti-human IgG antibody (Jackson, Pa., USA) in TBST (5% non-fat dry milkin Tris-buffered saline with 0.02% Tween 20) buffer. After washing withTBST, the blot was incubated with enhanced chemiluminescence reagents(Amersham Pharmacia Biotech) for visualization.

Example 5 Expression of Recombinant Receptor.Fc Fusion Protein

The extracellular domains of several innate immunity receptors fromimmune cells were cloned by reverse-transcription polymerase chainreaction (RT-PCR) according to the method of Example 1. The amplifiedDNA fragments were fused with the Fc portion of human IgG1 contained inthe pcDNA3/hIgG1-mutant plasmid. The cloned fusion genes was transfectedinto 293 FREESTYLE mammalian cells, and the secreted proteins werepurified by protein beads according to the method of Example 1. As shownin FIG. 1, sixteen C-type lectin genes were cloned (FIG. 1A).Specifically, FIG. 1A shows DNA fragments of innate immunity receptorsamplified by RT-PCR, then fractionated on 0.8% agarose and visualized byethidium bromide staining. FIG. 1B shows the expressed recombinantreceptor.Fc fusion proteins following electrophoresis on a 12% SDS-PAGEgel. In both FIG. 1A and FIG. 1B, the following lane designations areused: Lane 1: CLEC2B/AICL, Lane 2: CLEC4C/BDCA-2, Lane 3:CLEC13A/BIMLEC, Lane 4: CLEC1A/CLEC-1, Lane 5: CLEC4D/CLEC-6, Lane 6:CLEC12A/CLL-1, Lane 7: CLEC4A/DCIR, Lane 8: CLEC4L/DC-SIGN, Lane 9:CLEC4M/DC-SIGNR, Lane 10: CLEC7A/Detin-1, Lane 11: CLEC6A/Detin-2, Lane12: CLEC4H2/HBVxAgBP, Lane 13: CLEC4K/Langerin, Lane 14: KLRG/MAFAL,Lane 15: DLVR1/CLEC5A (MDL-1), Lane 16: CLEC4E/MINCLE. In addition, thehuman TREM (triggering receptor expressed on myeloid cells)-1, -2 andTREM-like transcripts (TLT)-1, -2 (Bouchon et al., 2000, J Immunol 164,4991-5; Daws et al., 2003, J Immunol 171, 594-9; Washington et al.,2002, Blood 100, 3822-4) were also cloned and expressed by similarstrategy.

Example 6 Dose-Dependent Interaction Between Immobilized PolysaccharidesWith Receptor.Fc Fusion Proteins

The interaction between polysaccharides and the receptor.Fc fusionproteins was tested using a dot-binding assay according to the method ofExample 4. The water soluble fraction 3 of Reishi polysaccharides (F3)(see Example 3) contains the active components to stimulate cellproducing cytokines (Wang et al., 2002, Bioorg Med Chem 10, 1057-62;Chen et al., 2004, Bioorg Med Chem 12, 5595-601; Chien et al., 2004,Bioorg Med Chem 12, 5603-9; Hsu et al., 2004, J Immunol 173, 5989-99).Reichi saccharide was known to contain either a polysaccharide backbonewith β-1, 3-linkages, or a polymannose backbone with α-1,4-linkage (Usuiet al., 1983, Carbohydr. Res., 273; Miyazaki and Nishijime, 1982,Carbohydr. Res. 109, 290; Bao et al., 2002, Phytochemistry 59, 175-81).The Dectin-1 receptor, a member of the C-type lectin family, has beenshown to interact with β-1,3-D-glycans (Brown and Gordon, 2001, Nature413, 36-7). Dectin-1 receptor has been shown to mediate the biologicaleffects of beta-glucans (Brown et al., 2003, J Exp Med 197, 1119-24).Thus the F3 portion of Reishi was tested to determine whether it couldinteract with the Dectin-1 receptor using the dot-binding assay ofExample 4.

Biotinylated F3 fraction (“Biotin-GLPS F3” in FIG. 2A) (preparedaccording to Example 2) was immobilized on a PVDF membrane after a5-fold serial dilution and incubated with streptavidin-conjugated HRP,and the resulting binding reaction was detected using enhancedchemiluminescence reagents (see Example 4). As shown in FIG. 2A, thesensitivity of this dot binding assay is better than about 0.08 μg. FIG.2A also shows that no background is seen when unbiotinylated F3(“GLPS-F3” in FIG. 2A) is immobilized on the PVDF membrane and thencontacted with streptavidin-conjugated HRP.

Un-biotinylated F3 fraction was also immobilized on a PVDF membraneafter serial dilution, and incubated with 100 μL of 1 μg/mL Dectin-1.Fcfusion protein or human IgG1 (as a negative control), followed byincubation with goat HRP-conjugated anti-human IgG (see Example 4). Asshown in FIG. 2B, Dectin-1.Fc can detect the presence of less than about1 ng of F3 in the dot-binding assay. There is no visible background onthe regions of the blot contacted with human IgG1 instead ofDectin-1.Fc.

The dot density of the blot of FIG. 2B was determined by a densitometer(ImageQuant), and the results show that the Dectin-1.Fc binding signalincreased in a dose-dependent manner (see FIG. 2C).

In order to determine whether other polysaccharides inhibit theinteraction between F3 and Dectin-1, F3 (lopg/dot) was immobilized onPVDF membrane and then contacted with 100 μL Dectin-1.Fc (1 μg/mL) inthe presence of serially diluted solutions of β-glucan, D-glucose, andD-galactose (0.1 μg-1000 μg), followed by incubation with goatHRP-conjugated anti-human IgG. FIG. 2D shows dot density analysis of theblot for competitor β-glucan, and FIG. 2E shows a blot image for all thecompetitors. It can be seen that the interaction between Dectin-1.Fc andthe F3 fraction is inhibited by β-1,3-glucan, but not by D-glucose orD-galactose. This indicates the interaction between Dectin-1.Fc with F3is via recognition of β-1,3-glucan.

Example 7 Identification of Receptors Capable of Interacting with F3Fraction

The interaction of F3 with other members of the C-type lectin family orwith Ig-like receptors was assayed. Non-biotinylated F3 andnon-biotinylated F3C (which is derived from F3 after passing through 100kDa MWCO centrifugal tube) (10 μg/dot) was immobilized on PVDF membrane(see Example 4), then incubated with 100 μL of 1 μg/mL solutions of 25different recombinant receptor.Fc fusion proteins (including 19 lectinreceptors, and 8 members of TREM and TLT families) and human IgG1 ascontrol. Binding was detected using goat HRP-conjugated anti-IgGantibody and ECL reagents. The results are depicted in table form inFIG. 3 (with relative dot intensities indicated by “+” symbols, and nodetectable binding indicated by “−” symbol) and an image of the blot isdepicted in FIG. 4A. The probe numbering system used in FIG. 3 isretained in FIG. 4A.

The results show that in addition to Dectin-1.Fc (probe no. 14 in FIG. 3and FIG. 4A), F3 also interacted with KCR.Fc (probe no. 7 in FIG. 3 andFIG. 4A), DC-SIGNR.Fc (probe no. 11 in FIG. 3 and FIG. 4A), and TLT-2.Fc(probe no. 21 in FIG. 3 and FIG. 4A). It is interesting to note thatF3C, which is derived from F3 after passing through 100 kDa MWCOcentrifugal tube, has less binding affinity to TLT2. This suggests thatTLT2 can differentiate the subtle difference between F3 and F3c.

Members of the lectin receptor family rely on Ca⁺⁺ for interaction;therefore, the ability of EDTA (Ethylene Diamine Tetra Acetic Acid) toinhibit binding to F3 was studied. It was found that EDTA (lomM in TBST)completely abolished the interaction of F3 with KCR.Fc and withDC-SIGNR.Fc, but not the interaction of F3 with Dectin-1.Fc and TLT2.Fc.FIG. 4B depicts images of the blots made in the presence and absence ofCa⁺⁺ (left panel is TBST only; right panel is 10 mM EDTA+TBST). Bindingwas detected using goat HRP-conjugated anti-IgG antibody and ECLreagents. This result agrees with previous observations that theinteraction between ligands and KCR (Hoyle and Hill, 1988, J Biol Chem263, 7487-92) and DC-SIGNR is Ca⁺⁺-dependent (Soilleux et al., 2000, JImmunol 165, 2937-42), while Ca⁺ is dispensable for the interactionbetween Dectin-1 and β-1,3-glucan (Herre et al., 2004, Mol Immunol 40,869-76). Thus, F3 apparently contains abundant glycans which caninteract with multiple receptors on immune cells simultaneously.

FIG. 4C depicts a dot blot using β-glucan as polysaccharide (10 μg/dot)and using 100 μL of 1 μg/mL Dectin-1.Fc, DC-SIGN.Fc, mKCR.Fc, andTLT2.Fc. Binding was detected using goat HRP-conjugated anti-IgGantibody and ECL reagents. Of the four receptor.Fc fusion proteinstested, only Dectin-1.Fc can bind to β1,3-glucan. This indicates thatthe other three receptor.Fc fusion proteins bind to sugar componentsother than β-1,3-glucan.

Example 8 Fingerprints of Polysaccharides from Various Sources

The dot-binding assay of Example 4 was performed using Dectini.Fc,mKCR.Fc, DC-SIGNR.Fc, and TLT2.Fc fusion proteins in order to obtain thefingerprints of polysaccharides isolated from Cordyceps and otherresources on market. Each polysaccharide composition was immobilized ona PVDF membrane as described above and then contacted with 100 L of a 11g/mL solution of the fusion protein. Binding was detected using goatHRP-conjugated anti-IgG antibody and ECL reagents. FIG. 5A shows theindividual dot blots for each fusion protein and FIG. 5B shows thesample key numbers and the relative dot intensities in table form. TheReishi crude extract (spot no. 5 in FIG. 5) only interacts withDectin-1.Fc and DC-SIGNR.Fc, while the purified F3 (spot no. 1) from thecrude extract interacts with all the four receptors. This indicates thatthe F3 purification process enriches the components that interact withimmune receptors. Polysaccharide from Cordyceps (spot no. 7) interactsstrongly with Dectin-1.Fc, indicating that the polysaccharide contains β1,3 glycan, but its interaction with the other three receptors is muchweaker than that of F3. Polysaccharides isolated from Dendrobiunhuoshanense test positive with the iodine test reaction (see Example 2)suggesting these fractions comprise mainly α-D-glucan. In contrast tothose isolated from fungi, the mixture of polysaccharides of D.huoshanense (spot no. 6) does not react with any of the four receptor.Fcfusion proteins. Polysaccharides isolated from mushroom polysaccharidesby ddH₂O (fraction L, spot no. 8) and 0.25N NaOH (fraction M, spot no.9) (see Example 2) bind differentially to Dectin-1.Fc and DC-SIGNR.Fc.Thus, this approach can produce distinct fingerprints frompolysaccharides isolated from different sources and preparations,

Examples 6-8 illustrate that F3 interacts with Dectin-1.Fc, mKCR.Fc,DC-SIGNR.Fc, and TLT2.Fc. The Kupffer cell receptor (KCR) has highaffinity to D-galactose and N-acetylgalactyosamine (Fadden et al., 2003,Glycobiology 13, 529-37), and is able to clear serum D-galactose- orD-fucose-terminated glycoprotein (Lehrman et al., 1986, J Biol Chem 261,7426-32). The immunomodulatory function of F3 is dependent on thepresence of fucose, and glycolytic cleavage by α1,2-fucosidase abolishesF3 activity. Thus it would be interesting to ask whether these fourreceptors can interact with F3 after glycolytic cleavage.DC-SIGNR/L-SIGN is structurally similar to DC-SIGN (77% identity), butit is only expressed in the endothelial cells of liver sinusoid, lymphnode and placenta (Van Liempt et al., 2004, J Biol Chem 279, 33161-7).Both DC-SIGN and DC-SIGNR can bind to N-linked high-mannoseoligosaccharides (Man₉ GlcNAc₂ Asn glycopeptide). However, only DC-SIGN,and not DC-SIGNR, can bind to glycans with a terminal fucose residue(Guo et al., 2004, Nat Struct Mol Biol 11, 591-8). Even though DC-SIGNRbinds relatively restricted ligands than DC-SIGN, only DC-SIGNR caninteract with F3. This suggests that F3 might contain a unique structuredistinct from Fucα1-4GlcNAc, Lewis^(X), Lewis^(a) and blood group sugarepitopes (the known ligands for DC-SIGN).

TLT-2 is a member of TREM-like transcripts family, which contain acharacteristic single V-set immunoglobulin (Ig) domain and a longcytoplasmic tail with a proline-rich region and an immune receptortyrosine-based inhibitory motif (ITIM), the latter known to be used forinteractions with protein tyrosine phosphatases (Washington et al.,2002, Blood 100, 3822-4; Washington et al., 2004, Blood 104, 1042-7).Since F3 has potent immunostimulatory functions, it would be interestingto study whether the removal of TLT2.Fc.binding components from F3 byaffinity chromatography could further enhance the stimulatory functionsof F3 in the future. Alternatively, F3 can be further purified byaffinity chromatography using Dectin-1.Fc, KCR.Fc, and DC-SIGNR.Fc toremove other components in F3.

The differential fingerprints between F3 and F3c; between F3 and Reishi1-3; and between mushroom polysaccharides fraction L and M, suggest thatthese four receptor.Fc fusion proteins exemplified herein can be used tooptimize purification procedures, and to monitor the variation ofpolysaccharides from different sources or from different fermentationconditions.

Example 9 Identification of Human Lectin Receptors that Interact withGLPS-F3 By Enzyme Linked Immunoassay on Microtiter Plates

The interactions of polysaccharides with receptor.Fc fusion proteins wasfurther investigated by performing an enzyme-linked immunoassay, whichwas based on immobilizing GLPS-F3 through both hydrophilic andhydrophobic forces onto microtiter plates (polysytrene). In this format,the number of different receptor.Fc fusions for profiling was increasedin comparison to Example 7. To optimize the quantity of GLPS-F3 forimmobilization, various amounts (3-1000 ng/well, diluted in loomM Trisbuffer, pH9.5) of biotinylated-GLPS-F3 (Biotin-GLPS-F3) were coated ontoMaxiSorp StarWell microtiter plates (50 μL/well; Nunc). The plates wereincubated overnight at 4° C., and then the wells were washed twice withTBST, followed by blocking with 200 μL blocking buffer (2% BSA/TBST) for1 hour at room temperature. Peroxidase-conjugated avidin (1:5000dilution, Vector Laboratories) and TMB (tetramethylbenzidine) substratewas then used for detection of immobilized biotinylated GLPS-F3. Asshown in FIG. 6A, the quantity of Biotin-GLPS-F3 for plate coatingreached plateau at 10 ng/well, which was therefore chosen to use forimmobilizing un-biotinylated GLPS-F3 in EIA.

The interaction between GLPS-F3 and receptor.Fc was then tested.Unbiotinylated GLPS-F3 was immobilized at 100 ng/well as describedabove, and 100 μL receptor.Fc fusion protein (1 μg/ml in 2 mM MgCl₂/2 mMCaCl₂/1% BSA/TBST) was added into each well and incubated for 1 hour atroom temperature. After washing with TBST, wells were incubated withperoxidase-conjugated goat anti-human IgG Ab (1:5000 dilution, JacksonImmunoResearch Laboratories) in blocking buffer at room temperature for30 min. Wells were incubated with 100 μL TMB substrate for 15 min afterTBST washing and read at 450 nm in a Fusion plate reader (Perkin Elmer).The results were normalized with respect to Fc.Dectin-1 binding(Dectin-1 is a known lectin receptor that binds to β-1,3-glucan which isthe backbone found in GLPS-F3). FIG. 6B depicts in graphical form theaffinity of each receptor for GLPS-F3 relative to Dectin-1. The resultsshow that high binding affinity to GLPS-F3 was observed for Fc.Langerin,Fc.DC-SIGN, MMR.Fc, TLR2.Fc, TLR4.Fc, Fc.CLEC-2 (CLECIB) and Fc.CLEC-6(CLEC4D) (high binding was defined in this assay as >50% bindingintensity compared to Fc.Dectin-1). It is noteworthy that TLR2 and TLR4,which have been demonstrated to play a role in GLPS-induced cellactivation (Hsu et al., J Immunol 173:5989-5999 (2004); Shao et al.,Biochem Biophys Res Commun 323:133-141 (2004)), bound to GLPS-F3 in theEIA format as well. There was also weaker but positive GLPS-F3 bindingability (25-50% binding intensity compared to Fc.dectin-1) found inFc.NKG2D, Fc.MINCLE, Fc.mKCR, DCAL1.Fc, DEC205.Fc, Endo180.Fc andNKp30(NCR3).Fc. Other lectin receptors including Fc.A1CL, Fc.BDCA2,Fc.CLEC1, Fc.CLL1, Fc.DCIR, Fc.DC-SIGNR, Fc.dectin-2, Fc.MDL-1 andFc.ML2 had minimal binding ability to GLPS-F3, as did control humanIgG1.

Example 10 Competition Assay For GLPS-F3-Interacting Innate ImmunityReceptors

To understand the interaction of GLPS-F3 with specific innate immunityreceptors, the polysaccharides mannan and β-glucan and themonosaccharides D-mannose (Man), D-glucose (Glc), N-acetyl-glucosamine(GlcNAc), D-galactose (Gal), N-acetyl-galactosamine (GalNAc), L-fucose(Fuc) and sialic acid, were used in a competition assay. Innate immunityreceptors that showed higher binding ability to GLPS-F3 were examined,including Fc.Dectin-1, Fc.Langerin, Fc.DC-SIGN, TLR4.Fc, MMR.Fc,Fc.CLEC-2 (CLECiB) and Fc.CLEC-6 (CLEC4D). The assays were carried outas in Example 9, with the addition of 1 mg/ml of each polysaccharide ormonosaccharide.

As shown in FIG. 7 (which shows graphically the % binding for eachreceptor/saccharide combination relative to the binding seen in theabsence of saccharide) and Table I (which provides the data from FIG. 7in tabular form), the interaction between GLPS-F3 and Fc.Dectin-1 couldbe blocked by β-glucan with 58% inhibition, which is in accordance withpublished results (Palma et al., J Biol Chem 281:5771-5779 (2006);Willment et al., J Biol Chem 276:43818-43823 (2001)). The addition ofsialic acid (83% inhibition) interfered with the binding of Fc.Dectin-1to GLPS-F3. The interaction between Fc.Langerin and GLPS-F3 wasdisrupted by mannan, Man and GlcNAc (95%, 26% and 84% inhibition), whichare reported as the sugar ligands for Langerin (Stambach & Taylor,Glycobiology 13:401-410 (2002)); sialic acid (95% inhibition) was alsoobserved to interfere with the binding of Fc.Langerin to GLPS-F3. As forthe binding of Fc.DC-SIGN to GLPS-F3, mannan, Man, Fuc and sialic acidshowed a potent blocking activity (98%, 72%, 92% and 90% inhibition),while Glc and GlcNAc had a weaker effect (45% and 27% inhibition,respectively) in blocking the interaction. Mannan, Man, Glc, GlcNAc,Gal, Fuc and sialic acid blocked the interaction (98%, 87%, 45%, 78%,36%, 88% and 93% inhibition) between GLPS-F3 and MMR.Fc, an importantlectin receptor that is known to bind Man, Fuc, GlcNAc and sialyl Lewisx (sLex) (Letuex et al., J Exp Med 191:1117-1126 (2000); Stahl, Am JRespir Cell Mol Biol 2:317-318 (1990)). The interaction of Fc.CLEC-2 toGLPS-F3 was blocked by the addition of sialic acid (55% inhibition). ForFc.CLEC-6, no obvious blocking was observed among the sugar tested.Notably, mannan and Fuc showed a blocking effect (72% and 44%inhibition, respectively) on TLR4.Fc and GLPS-F3 interaction: The dataobtained here was in line with the results reported by the study ofsugar ligands for Dectin-1, Langerin, DC-SIGN and MMR. It was alsoindicated that many lectin receptors could bind to GLPS-F3 withmultivalency through different sugar components.

TABLE 1 Percentage of binding of innate immunity receptor. Fc fusions toGLPS- F3 in the presence of sugar competitors relative to binding seenin absence of sugar competitor. Innate Immunity Receptor Sugar Dectin-1Langerin DC-SIGN TLR4.Fc MMR.Fc CLEC-2 CLEC-6 none 100 ± 7.6  100 ± 1.0 100 ± 0.1  100 ± 4.5  100 ± 2.2  100 ± 8.0  100 ± 0.8  mannan 82 ± 0.2 5 ± 0.9  2 ± 0.5 28 ± 6.3  2 ± 0.5 88 ± 6.3 75 ± 0.8 Man 89 ± 0.5 74 ±2.1 28 ± 1.3 89 ± 2.6 13 ± 6.4 95 ± 9.1 98 ± 3.7 b-glucan 42 ± 0.3 77 ±3.2 81 ± 1.4 96 ± 4.0 100 ± 4.2  95 ± 5.1 98 ± 7.3 Glc 86 ± 1.5 87 ± 2.055 ± 5.4 101 ± 4.7  55 ± 2.9 108 ± 6.8  100 ± 5.3  GlcNAc 91 ± 2.6 16 ±2.7 73 ± 8.6 99 ± 4.7 22 ± 3.4 103 ± 10.2 99 ± 8.5 Gal 88 ± 0.4 92 ± 0.982 ± 4.0 100 ± 2.7  64 ± 4.2 104 ± 5.1  90 ± 3.7 GalNAc 88 ± 2.6 97 ±0.7 110 ± 3.1  95 ± 4.1 80 ± 4.2 110 ± 11.4 96 ± 2.4 Fuc 92 ± 5.2 76 ±1.9  8 ± 1.8 56 ± 1.9 12 ± 2.0 91 ± 8.5 82 ± 6.5 sialic acid 17 ± 0.3  5± 0.3 10 ± 1.0 77 ± 3.1  7 ± 2.4 45 ± 6.2  94 ± 13.0

The systems presented in Examples 7-10 are useful tools for highthroughput profiling of not only GLPS, but also other glycoproteinmixtures including many Chinese herb drugs currently in use. By usingdifferent surfaces for immobilizing polysaccharides (PVDF andpolystyrene), different profiles were obtained for GLPS-F3. This may bedue to preferential binding of certain polysaccharides within themixtures to different surfaces. The results obtained from these twocomplementary formats provide “fingerprints” of polysaccharide mixtures.These strategies of fingerprinting polysaccharide mixtures can be used,for example, to monitor the contents of herb extracts under differentconditions, from different sources, or from different batches. Moreover,the information gathered from the profiles of specific polysaccharidemixtures will be of great importance in understanding the underlyingmolecular mechanisms of their biological effects in vivo.

Example 11 Detection of the Interaction of DVLR1/CLEC5A (MDL-1) withDengue Virus

The following examples show how the fusion proteins and methods of thedisclosure can be used to identify the innate immunity receptor(s) thatinteract with a pathogen, and how that information can subsequently beused to determine the downstream effects of pathogen binding to theinnate immunity receptor, and also to design therapeutic agents for thetreatment of pathogen infection.

Dengue is one of the most important mosquito-borne viral diseaseaffecting humans. Its global distribution is comparable to that ofmalaria, and an estimated 2.5 billion people live in areas at risk forepidemic transmission. The clinical syndromes after dengue virus (DV)infection include dengue fever (DF) and dengue hemorrhagic fever(DHF)/dengue shock syndrome (DSS). However, the underlying molecularmechanisms leading to DHF and DSS are still not well elucidated.

DC-SIGN is known to mediate DV infection of human dendritic cells(Tassaneetrithep et al., J Exp Med, 2003. 197(7): p. 823-9). In order tounderstand the pathogenesis of DV, it is important to determine whetherDV can interact with other membrane-bound C-type lectin receptors andC-type-like lectin receptors from dendritic cells, macrophages, naturalkiller cells, and peripheral blood mononuclear cells (PBMCs). To thisend, the extracellular domains of DVLR1/CLEC5A (MDL-1), Dectin-1, KCR,and DC-SIGN (as a positive control) were fused to the Fc portion ofhuman IgG1. Specifically, primers for DC-SIGN (SEQ ID NO: 17 and SEQ IDNO:18), DVLR1/CLEC5A (SEQ ID NO: 21 and SEQ ID NO:22), Dectin-1 (SEQ IDNO:25 and SEQ ID NO:26) and KCR (forward: 5′-CAGCCTTGGAGACCTGAGT-3′ SEQID NO: 37; reverse 5′-TAGCCTACTCTGGCCGC-3′ SEQ ID NO:38) were used togenerate amplified cDNA fragments. Each forward primer had an extraBamH1 site, and each reverse primer had an extra EcoRI site tofacilitate the subcloning of the amplified cDNA into the pcDNA3.1(Invitrogen) mammalian expression vector containing the human IgG1 Fcportion. The resulting vector was then transfected into 293 FreeStylecells (Invitrogen) to produce soluble recombinant proteins. Allrecombinant receptor.Fc fusion proteins were purified by protein ASepharose beads (Pharmacia) and eluted with 0.1M glycine-HCl (pH0.3).

One μg of each receptor.Fc fusion protein was coated onto microtiterplates overnight at 4° C. DV (5×10⁶ particles) of strain 16681 (a DEN2strain) in binding buffer (1% BSA, 2 mM CaCl₂, 2 mM MgCl₂, 50 mMTris-HCl pH 7.5, 150 mM NaCl) was then added to the plates and theplates were incubated for 2 hours. After washing non-bound virus, abiotinylated anti-DEN2 envelope protein antibody (Wu et al., J Virol,2002. 76(8): p. 3596-604) was applied to bind to the virus for 1 hour.Diluted horseradish peroxidase-conjugated streptavidin was then added tothe plates, followed by a 1 hour incubation. TMB substrate was thenadded and the plates were read using an ELISA reader at OD450 nm.

The results are depicted in FIG. 8A (** indicates p<0.01, *** indicatesp<0.001 (Student's t test)). The results show that in addition toDC-SIGN (positive control), DV also binds to DVLR1/CLEC5A. To confirmthis result, immunoprecipitation studies were performed with human IgG1(negative control), DC-SIGN.Fc, KCR.Fc, and DVLR1/CLEC5A.Fc.Specifically, 5×10⁶ Dengue virus particles were incubated with 5 μg ofeach protein, and then Protein A beads were added. The resultingimmunocomplexes were washed, separated by SDS-PAGE, and transferred ontonitrocellulose membrane. The membrane was then probed with biotinylatedanti-DEN2 envelope protein antibody and developed with horseradishperoxidase-conjugated streptavidin. The results are shown in FIG. 8B.The results show that only DC-SIGN.Fc and DVLR1/CLEC5A.Fc were able toimmunoprecipitate DV.

The microtiter plate assay was repeated in the presence of EDTA (10 mM)to chelate Ca⁺⁺ cations. The results (FIG. 8C) reveal that DVLR1/CLEC5Abinding to Dengue virus is Ca⁺⁺ independent, whereas DC-SIGN binding isCa⁺⁺ dependent (*** indicates p<0.001, Student's t test).

The microtiter plate assay was also repeated for DVLR1/CLEC5A.Fc fusionprotein with DV particles (5×10⁶) that had been 1) preincubated with 500U of the glycosidase PNGaseF (New England Biolabs, Inc.) overnight at37° C.; or 2) treated with dithiothreitol (DTTI) (0.1M); or 3) incubatedat 95° C. for 5 minutes; or 4) UV irradiated for 5 minutes. The resultsare shown in FIG. 8D (asterisks indicate where the binding affinity ofDVLR1/CLEC5A.Fc fusion protein is altered by modification of the virusrelative to non-treated virus; ** p<0.01, *** p<0.001, Student's ttest). The results indicate that pretreatment of DV with PNGase Finhibited DVLR1/CLEC5A.Fc interaction significantly, and thatpretreatment with either heat or dithiothreitol almost completelyinhibited DVLR1/CLEC5A.Fc binding, but not DC-SIGN.Fc binding to DV.This suggests that both the sugar epitope(s) and the three dimensionalconformation of DV are important for binding to DVLR1/CLEC5A.

To evaluate the expression of DVLR1/CLEC5A on immune cells, flowcytometric analysis was performed on human polymorphonuclear (PMN) cells(neutrophils), PBMCs, macrophages, and dendritic cells. PMNs and PBMCswere isolated from the whole blood of human healthy donors by dextransedimentation as described (Kuan et al., Br. J. Pharmacol., 2005,145(4):460-468) and standard density gradient centrifugation withFicoll-Paque respectively (Amersham Biosciences, Piscataway, N.J.).Purified neutrophils were resuspended in phosphate saline buffer (PBS,pH 7.4,) with hypotonic lysis of erythrocytes. CD14+ cells weresubsequently purified from PBMCs by high-gradient magnetic sorting usingthe VARIOMACS technique with anti-CD 14 microbeads (Miltenyi BiotecGmbH, Bergisch Gladbach, Germany), then were cultured in completeRPMI-1640 medium (Life Technologies, Gaithersburg, Md.) supplementedwith ng/ml human M-CSF (R&D Systems, Minneapolis, Minn.) for 6 days(Chang et al., J. Leukoc Biol, 2004, 75(3):486-494). Dendritic cells(DC) were generated from adherent PBMCs by culture in RPMI 1640 mediumsupplemented with 10% fetal calf serus, 800 U/ml human GM-CSF (Leucomax;Schering-Plough, Kenilworth, N.J.), and 500 U/ml human IL-4 (R&DSystems) for 6 days (immature DCs). To prepare mature activated DCs,immature DCs were further incubated with gamma-irradiated (5500 rad)CD40 ligand (CD40L)-expressing L cells (DNAX Research Institute, PaloAlto, Calif.) at a ratio of 3:1 for 36 hr (Hsu et al., J. Immunol.,2002, 168(10):4846-4853).

Flow cytometry was performed on the above-mentioned cell types usingFITC-conjugated anti-DVLR1/CLEC5A monoclonal antibodies (R&D Systems,Minneapolis, Minn.), or FITC-conjugated anti-DC-SIGN monoclonalantibodies (ED PharMingen), in conjunction with Phycoerythrin(PE)-conjugated anti-CD3, CD19, CD56, CD14, and CD66 antibodies fordouble staining (BD PharMingen). Matched isotype controls (IgG2b forDVLR1 mAb, IgG 1 for DC-SIGN; Sigma) were also performed in this surfacestaining to provide background information. Fluorescence was analyzed byFACSCalibur flow cytometry (Becton Dickinson) with CellQuest software(Becton Dickinson). CD marker positive cells were gated to determine theexpression of DVLR1/CLEC5A or DC-SIGN. The results are shown in FIG. 9A(DVLR1/CLEC5A) and FIG. 9B (DC-SIGN) (shaded area represents isotypecontrol). The results indicate that DC-SIGN is mainly expressed onimmature dendritic cells, and is weakly expressed on macrophages. Theresults also indicate that DVLR1/CLEC5A was detected on the surface ofCD14+ derived macrophages (M), CD66+ PMNs and CD14+ freshly isolatedPBMCs, but not on CD14+ derived immature and mature dendritic cells.This is in accord with previous observations that DVLR1/CLEC5A mRNA isexpressed in human monocytes and macrophages, but not in dendritic cells(Bakker et al., Proc. Natl. Acad Sci USA, 1999, 96(17):9792-9796).

The results presented in this example show that the receptor.Fc fusionprotein-based methods disclosed herein can be used to determine theidentity of the innate immunity receptors that bind to a specificpathogen, such as Dengue virus. This in turn allows one to identify thecell types that interact with the pathogen, and furthermore provides anew target for treatment or prevention of infection by the pathogen. Forexample, the results disclosed herein suggest that agents that preventDV from binding to DVLR1/CLEC5A can be used for prophylactic ortherapeutic purposes. For example, monoclonal antibodies againstDVLR1/CLEC5A can be generated by one skilled in the art that prevent thebinding of DV to DVLR1/CLEC5A. Moreover, since DV is a member of thefamily Flaviviridae, this result suggests that DVLR1/CLEC5A may interactwith other viruses within the same family, for example, viruses withinthe genus Flavivirus (such as West Nile Virus, Japanese encephamyelitisvirus (JEV), yellow fever virus, tick-borne encephamyelitis virus) andviruses within the genus Hepacivirus (such as Hepatitis C virus).Accordingly, DVLR1/CLEC5A may serve as a therapeutic or prophylactictarget for these viruses also. In addition, since DVLR1/CLEC5A is apattern recognition receptor, DVLR1/CLEC5A may serve as a therapeutic orprophylactic target for other enveloped viruses.

Example 12 Dengue Virus Induced DAP12 Phosphorylation is Mediated ViaDVLR1/CLEC5A

DVLR1/CLEC5A (MDL-1) is a type II transmembrane protein comprising 187aa in length, and it includes a charged residue in the transmembraneregion that enables it to pair with DAP12 (DNAX activating protein of 12kDa) (Bakker et al., Proc. Natl. Acad Sci USA, 1999, 96(17):9792-9796).DAP12 is a disulfide-linked, homodimeric transmembrane protein with aminimal extracellular domain, a charged aspartic acid in thetransmembrane domain and an ITAM (immunoreceptor tyrosine-basedactivation motif) in its cytoplasmic tail. Because DV binds toDVLR1/CLEC5A on CD14+ macrophages, and because DAP12 has an ITAM, it wasof interest to determine whether DV can induce DAP12 phosphorylation inCD14+ macrophages. Accordingly, CD14+ macrophages were infected with DVusing the a slight modification of the method disclosed in Chen et al,J. Virol. 2002, 76(19):9877-9887. Briefly, terminal differentiatedmacrophages were washed once with incomplete RPMI medium to remove fetalcalf serum in culture medium. The cells were then infected with DV atdifferent multiplicities of infection (MOI). The virus was incubatedwith the cells in serum-free RPMI at 37° C. for 2.5 h to permit viraladsorption. The culture plates were gently agitated every 30 min foroptimal virus-cell contact. Thereafter, the unabsorbed viruses wereremoved by washing the cell monolayers twice with serum-free RPMI andthen once with incubation, the cell-free supernatants were harvestedseparately and stored in aliquots at −80° C. until assayed forinfectious-virus production and cytokine secretion (see Example 13).Infectious virus titers were determined by a plaque forming assay onBHK-21 cells. Plaques were counted by visual inspection at 7 days aftercrystal violet overlay to determine the number of plaque-forming units(PFU) per mL of supernant (Lin et al., J. Virol., 1998,72(12):9.729-9737). To detect intracellular DV antigens, infected cellswere fixed with 1% paraformaldehyde and permeabilized with 0.1% saponin,followed by staining with NS3 mAb (Lin et al., J. Virol., 1998,72(12):9729-9737) or matched isotype control (IgG1; Sigma). Afterincubation for 1 h, PE-conjugated goat F(ab)′ anti-mouse IgG secondarywas added for fluorescence detection and fluorescence was analyzed byFACSCalibur flow cytometry with CellQuest software.

The results are shown in FIG. 10A-D. At 48 h after infection at MOI=5,DV non-structural protein 3 (NS3) was detected by flow cytometry in thecytosol of macrophages (FIG. 10A; gray histogram is antibody isotypecontrol). The extracellular virus titer was measured at various timesfollowing infection, and revealed that virus particles were released toculture supernatant when macrophages were infected with live DV, but notwith UV-irradiated DV (UV-DV; 254 nm irradiation for 15 minutes on iceat 5 to 10 cm distance) (FIG. 10B).

DAP12 phosphorylation was studied 2 hours after infection at varyingMOIs (MOI=0.05-30, 2 h after infection), and also at a fixed MOI (MOI=5)over a time course (2-48 h after infection). Specifically, for detectionof phospho-DAP12, macrophages were stimulated with DV for theappropriate amount of time at the appropriate MOI and then lysed inlysis buffer (50 mM Tris-HCl [pH7.5], 150 mM NaCl, 1% Triton X-100, 0.1%SDS, 5 mM EPTA, 10 mM NaF, 1 mM sodium orthovanadate, and proteinaseinhibitor cocktail tablet [Roche]). Equal amount of total cell extractswere immunoprecipitated with DAP12 rabbit polyclonal antibody (SantaCruz Biotechnology Inc, CA) and protein A sepharose (AmershamBiosciences AB) for 4 h at 4° C. After incubation, the immunocomplex waswashed three times and separated by SDS-PAGE, followed by transferringonto nitrocellulose membrane and probed with anti-phosphotyrosineantibody (4G10; Upstate Biotechnology, Inc). Immunoblots were developedusing HRP-conjugated second antibody and enhanced chemiluminescence(Amersham). For reprobing, the membrane was stripped with a strongre-probe kit (Chemicon) and blotted with DAP12 antibody.

The results obtained at various MOIs are shown in FIG. 10C, and the timecourse experiment results are shown in FIG. 10D. The results show thatat 2 h after DV infection, the intensity of DAP12 phosphorylationincreased as the MOI was raised from MOI=0.05, reaching a peak whenMOI=(FIG. 10C). DAP12 phosphorylation was detected at 2 h after DVinfection, peaked at 12 h, and lasted for at least 48 h (FIG. 10D). Eventhough UV-DV could not replicate in CD14+ macrophages and had noactivity in a plaque assay (FIG. 10B), DAP12 was also phosphorylated at2 h and phosphorylated DAP12 remained detectable at 12 h, even thoughthe intensity is much weaker than that induced by live DV (FIG. 10D;UV-DV). This suggests that DV-induced DAP12 phosphorylation has twophases: phase I (in the first 6 h) is replication-independent, whilephase II (after 12 h) is replication-dependent.

To confirm that DAP12 phosphorylation was via DVLR1/CLEC5A, RNAinterference (RNAi) with short hairpin RNA (shRNA) was used to inhibitthe expression of DVLR1/CLEC5A in CD14+ macrophages and DAP12phosphorylation was assayed as above. Specifically, the coding region ofhuman DVLR1/CLEC5A was targeted with the following DVLR1/CLEC5A siRNA:

SEQ ID NO: 39 5′-TTGTTGGAATGACCTTAT-3′This stretch was adapted with loop sequence (TTCAAGAGA) from Brummelkampet al., Science, 2002, 296(5567): 550-553, to create an shRNA. Thepolymerase III terminator stretch used here was TTTTTT. The shRNA wascloned into the pLL3.7 gene silencing vector (Rubinson et al., Nat.Genet., 2003, 33(3):401-406) which contained loxP sites, a CMV(cytomegalovirus) promoter driving expression of enhanced greenfluorescent protein (EGFP), and a U6 promoter with downstreamrestriction sites (HpaI and XhoI). A DC-SIGN shRNA construct was alsoconstructed by subcloning the shRNA contained in the constructpSUPER-siDC-SIGN (Tassaneetrithep et al., supra) into pLL3.7 vectordigested with HpaI/XhoI. The constructs were electroporated intomacrophages using the Amaxa kit (Gaithersburg, Md.) accordingmanufacturer's specifications. Briefly, macrophages (6×10⁶) wereharvested as described above and resuspended in 100 μL of nucleofactorsolution. After the addition of siRNA (5 μg) or vector control, cellswere electroporated using Amaxa program Y-001 and allowed to recover for16 h. The efficiency of DVLR1 and DC-SIGN silencing was analyzed 24 hrsafter transfection by immunoblotting using anti-DVLR1/CLEC5A and DC-SIGNmonoclonal antibodies (R&D Systems), respectively.

The results are shown in FIG. 11. CD14+ macrophages electroporated withthe control vector pLL3.7 or with DC-SIGN-shRNA did not show a reductionin DAP12 phosphorylation after DV infection. By contrast, DAP12phosphorylation decreased dramatically in CD14+ macrophageselectroporated with DVLR1/CLEC5A-shRNA prior to DV infection. Therefore,it was concluded that DV-induced DAP12 phosphorylation occurs viaDVLR1/CLEC5A.

Example 13 DVLR1/CLEC5A is Involved in DV-Mediated TNF-α Release, Butnot Entry to CD14+Macrophages

Upon DV infection, CD14+ macrophages secrete pro-inflammatory cytokinesand chemokines, including tumor necrosis factor alpha (TNF-α),alpha-interferon (IFN-α), MIP-1α, and IL-8 (Chen et al, supra). Thelevels of TNF-α in culture supernatant were measured in DV-infectedCD14+ macrophages using a commercial ELISA kit. Measurements were madeat different MOIs and at different times post-infection for both live DVand UV-DV. The results are shown in FIG. 12A-C (error bars represent thestandard error from the mean of triplicates, and asterisks indicatestatistically different levels of cytokine production, *=p<0.05;**=p<0.01; ***=p<0.001). The results show that at 6 hours postinfection, both live DV and DV-UV had similar effects on TNF-α secretionat MOIs ranging from 0.05-30 (FIG. 12A). At 12 hours post infection,TNF-α secretion increased in a dose dependent (increasing MOI) manneronly for live DV. For UV-DV infected cells at 12 hours post infection,TNF-α levels remained the same at all MOIs (FIG. 12B). FIG. 12C shows atime course measurement of TNF. The results show that when infected withlive DV at MOI=5, TNF-α secretion increased rapidly from 6 h (8 μg/ml)to 12 h (85 μg/ml), and peaked at 48 h (350 μg/ml). When incubated withUV-DV, however, TNF-α secretion decreased from 8 μg/ml (at 6 h) to 5μg/ml (at 12 h). This suggests that the initial response (at 6 h) isindependent of virus replication, while the later phase of TNF-αsecretion (after 12 h) correlates with DV replication.

DC-SIGN has previously been shown to interact with DV in order tomediate virus entry into dendritic cells. Using the RNAi methodology andreagents of the prior examples, the effect of DC-SIGN-shRNA andDVLR1/CLEC5A-shRNA on NS3 expression in DV-infected CD14+ macrophageswas investigated. FIG. 13A shows that DC-SIGN-shRNA andDVLR1/CLEC5A-shRNA can knock down their respective proteins (pWTSI andpLL3.7 are no insert controls). FIG. 13B depicts the results of flowcytometry analysis and illustrates that only DC-SIGN-shRNA couldattenuate DV NS3 expression in CD14+ macrophages. This result wasconfirmed using immunofluorescence confocal microscopy using anti-DS3antibodies. FIG. 13C illustrates real time PCR analysis of virus titerin the supernatant of cells electroporated with the shRNA constructs.The results indicate that only DC-SIGN-shRNA is capable of reducingvirus titer in the supernatant of DV-infected cells.

Example 14 DVLR1/CLEC5A is Involved in DV-Induced ProinflammatoryCytokine Release from CD14+ Macrophages

The cytokine release profile for CD14+ macrophages infected with DV(MOI=5) was evaluated using ELISA after knock down of DVLR1/CLEC5A andDC-SIGN according to the methods of the preceding examples (2.5 htransfection). In the first 12 h, DC-SIGN-shRNA did not affect thesecretion of TNF-α, MIP-1α, IFN-α, IL-6, or IL-8. See FIG. 14A-B (errorbars represent the standard error from the mean of triplicates, andasterisks indicate statistically significant differences compared tocontrol experiments; *=p<0.05; **=p<0.01; ***=p<0.001). After 48 h,DC-SIGN-shRNA had a mild inhibitory effect (less than 20%) on TNF-α,MIP-1α, IFN-α, and IL-6 secretion; IL-8 secretion was not affected.Since DC-SIGN is involved in virus entry and replication, thisobservation suggests that initial cytokine secretion (first 12 h) isindependent of DV replication. In contrast, knock down of DVLR1/CLEC5Adramatically suppressed (p<0.005) the secretion of TNF-α, MIP-1α, IFN-α,IL-8, but not of IL-6. This suggests that DVLR1/CLEC5A is responsiblefor DV-induced cytokine release from CD14+ macrophages. Accordingly,therapeutic agents that prevent the binding of DV to DVLR1/CLEC5A willbe useful for preventing the deleterious effects of DV-induced cytokinerelease in humans. For example, monoclonal antibodies that preventDVLR1/CLEC5A interaction with DV will be useful for preventing ortreating DV-induced Dengue shock syndrome (DSS) or Dengue haemorrhagicfever (DHF).

Example 15 Antagonistic Anti-DVLR1/CLEC5A Monoclonal Antibodies (mAbs)Abolish Inflammatory Cytokine Release By DV Serotypes 1,2,3, and 4

Monoclonal antibodies against DVLR1/CLEC5A were generated using standardtechniques. Briefly, mice were immunized with DVLR-1.Fc fusion protein,and hybridomas were formed by fusing spienocytes from the mice withP3/NSI/1-Ag4-1 [NS-1] myeloma cells (ATCC TIB-18). Among the mAbsgenerated, clone 9B12, the subclones of 3E12 (clones 3E12A2, 3E12C1,3E12G9), and clone 8H8F5 suppressed TNF-α release from macrophages afterinfection with DEN1 (strain 766733A), DEN2 (strain PL046), DEN3 (strainH-87), and DEN4 (strain 866146A) in a dose-dependent manner. See FIG. 15which shows ELISA measurements of TNF-α secreted into culturesupernatants by CD14+ macrophages infected with DV. In accordance withstandard nomenclature, each antibody is referred to via the clone numberof the hybridoma that secretes it. Hence, the disclosure also providesthe hybridomas that secrete the abovementioned monoclonal antibodies.

The results demonstrate that anti-DVLR1/CLEC5A antibodies will serve asuseful therapeutic agents for preventing proinflammatory cytokinerelease from DV-infected CD14+ macrophages in humans. In particular, butnot exclusively, the monoclonal antibodies of this Example, or fragmentsthereof, or antibodies (or fragments thereof) that bind to the sameepitopes as the antibodies of this Example, may be formulated aspharmaceutical compositions and then administered for the treatment orprophylaxis of DV infection in humans, according to the methods providedherein.

Example 16 Determination of Pattern Recognization Receptors (PRRs) onImmune Cells that are Activated by Dengue Virus

Dendritic cells (DCs) and macrophages are the primary targets of DVinfections (Halstead et al., J. Exp. Med. 1977, 146:201-217; Palucka,Nat. Med. 2000, 6:748-749; Wu et al., Nat. Med. 2000, 6:816-820). Whileinfected DCs undergo apoptosis (despite the secretion of proinflammatorycytokines by bystander DCs) (Palmer et al., J. Virol. 2005, 79, 24322439), infected macrophages survive for at least 45 days and secretemultiple cytokines and chemokines from 6 h post-infection (Chen et al.,J. Virol. 2002, 76:9877-9887). This suggests that macrophages are themajor source of proinflammatory cytokines following DV infection, wherevirions might trigger inflammatory reactions by activating patternrecognition receptors (PRRs). Toll-like receptors (TLRs), C-type lectinsand immunoglobulin-like (Ig-like) receptors (e.g., TREMs and TREM-likereceptors (TLT)) have been implicated as potential PRRs (Cook et al.,Nat. Immunol. 2004, 5, 975 979; Klesney-Tait et al., Nat. Immunol. 2006,7, 1266 1273; Robinson et al., Nat. Immunol. 2006, 7, 1258 1265) in thiscontext.

To determine whether dengue virus binds to and activates candidate PRRson immune cells, twenty-two fusion proteins were expressed in mammaliancells and screened for their interaction with DV2 (Table 2). The fusionproteins comprised the human IgG1.Fc fragment in combination with theextracellular domains of C-type lectins and Ig-like receptors. GeneAliase Forward primer Reverse primer

TABLE 2 Construction of recombinant receptor.Fc fusion proteins. GeneSymbol Aliases Forward primer Reverse primer CLEC1A CLEC1GGATCCTTTCAGTACTACCAGCTCTCC GAATTCTCAGTCACCTTCGCCTAATGT (SEQ ID NO: 40)(SEQ ID NO: 2) CLEC1B CLEC2 GGATCCCTGGGGATTTGGTCTGTCGAATTCTTAAGGTAGTTGGTCCAC (SEQ ID NO: 3) (SEQ ID NO: 4) CLEC2B AICLGGATCCTCTCAGAGTTTATGCCCC GGATCCCCCCATTATCTTAGACAT (SEQ ID NO: 5)(SEQ ID NO: 6) CLEC4A DCIR GGATCCTTTCAAAAATATTCTCAGCTTCTTGAATTCTCATAAGTGGATCTTCATCATC (SEQ ID NO: 7) (SEQ ID NO: 8) CLEC4C BDCA2GGATCCTTTATGTATAGCAAAACTGTCAAG GAATTCTTATATGTAGATCTTCTTCATCTT(SEQ ID NO: 9) (SEQ ID NO: 10) CLEC4D CLEC6 GGATCCCATCACAACTTTTCACGCTGTGAATTCCTAGTTCAATGTTGTTCCAGG (SEQ ID NO: 41) (SEQ ID NO: 12) CLEC4EMincle GAAGATCTACATTTCGCATCTTTCAAACC GAATTCCTAGTTCAATGTTGTTCCAGG(SEQ ID NO: 13) (SEQ ID NO: 42) CLEC4F KCLR AGATCTACAGCAGACAAGACCTGAGATCTAGCGCCAGGAGCCCTCTG (SEQ ID NO: 43) (SEQ ID NO: 44) CLEC4K LangerinGGATCCCGGTTTATGGGCACCATA GGATCCTCACGGTTCTGATGGGAC (SEQ ID NO: 17)(SEQ ID NO: 18) CLEC4L DC- GGATCCAAGGTCCCCAGCTCCATAAGGAATTCCTACGCAGGAGGGGGGT SIGN (SEQ ID NO: 19) (SEQ ID NO: 20) CLEC4M DC-GGATCCAAGGTCCCCAGCTCCCTAA GAATTCCTATTCGTCTCTGAAGCAGG SIGNR(SEQ ID NO: 45) (SEQ ID NO: 22) DLVR1/ MDL-1 AGATCTAGTAACGATGGTTTCACCACGAATTCCTGTGATCATTTGGCATTCTT CLEC5A (SEQ ID NO: 23) (SEQ ID NO: 24)CLEC6A Dectin- GGATCCACATATGGTGAAACTGGC GGATCCAGCTTCTACTCATAGGTA 2(SEQ ID NO: 25) (SEQ ID NO: 26) CLEC7A Dectin-GGATCCACCATGGCTATTTGGAGATCC GAATTCTTACATTGAAAACTTCTTCTCAC 1(SEQ ID NO: 27) (SEQ ID NO: 46) CLEC10A HML2 GGATCCTCCAAATTTCAGAGGGACCTGGAATTCTCAGTGACTCTCCTGGCTG (SEQ ID NO: 29) (SEQ ID NO: 30) CLEC12A CLL-1GGATCCGTAACTTTGAAGATAGAAATGAAA GAATTCTCATGCCTCCCTAAAATATGTA(SEQ ID NO: 31) (SEQ ID NO: 47) CLEC13B DEC-205 GCCCAGTGCACCTACTATAGGTGCACTGGGCCTGTCTGGGTCC (SEQ ID NO: 48) (SEQ ID NO: 49) NKG2DGGAGTGCTGTATTCCTAAAC GAATTCCTGGCTTTTATTGAGATGG (SEQ ID NO: 50)(SEQ ID NO: 51) TREM1 GAAGGATGAGGAAGACCAGGC CATCGGCAGTTGACTTGGGTG(SEQ ID NO: 52) (SEQ ID NO: 53) TREM2 AGGGTGGCATGGAGCCTCTCGAATTCCACATGGGCATCCTCGAA (SEQ ID NO: 54) (SEQ ID NO: 55) TLT1CAGCCATGGGCCTCACCCTG GAATTCCTGGCTGGGTTCCAAAGGG (SEQ ID NO: 56)(SEQ ID NO: 57) TLT2 GAATTCCTGGCTGGGTTCCAAAGGG GAATTCCTGGTGCCTGATGGAGGGC(SEQ ID NO: 58) (SEQ ID NO: 59)

As shown in FIG. 16, DV interacts with DLVR1/CLEC5A. Specifically, FIG.16 a shows the interaction of DV (5×10⁶ PFU) with receptor.Fc (1 μg)determined by ELISA. In FIG. 16 b, complexes of DV (5×10⁶ PFU) withreceptor.Fc (5 μg) were immunoprecipitated and detected on westerns withmAb to DV envelope (E) protein. FIG. 16 c shows the inhibition ofDLVR1/CLEC5A-DV interaction by EDTA (10 mM) as determined by ELISA.FIGS. 16 d and 16 e show sugar competition assays wherein both DC-SIGN(CLEC4L) and DLVR1/CLEC5A increase DV binding to human 293T cells (FIG.16 d), while addition of sugars inhibits biotinylated DV binding toDC-SIGN- (left panel) or DLVR1/CLEC5A- (right panel) transfected 293Tcells in a dose-dependent manner as determined by flow cytometry (FIG.16 e). According to FIG. 16 e, MFI represents mean fluorescenceintensity. Units (U) for monosaccharide (mannose and fucose) andpolysaccharide (mannan) are mM and mg/ml, respectively. FIG. 16 f showsthe effect of PNGaseF (500 U), DTT (0.1 M), heat (95° C. 5 min), or L UV(10 J/cm²) on DLVR1/CLEC5A-DV interaction as measured by ELISA. Data areexpressed as the mean±s.d. of three independent experiments. Two-tailed,Student's t-tests were performed.

Among the receptors tested, DC-SIGN has been shown previously tointeract with glycans located on the envelope (E) protein of DV(Pokidysheva, E. et al., Cell 124:485-93 (2006)). Using ELISA,DLVR1/CLEC5A.Fc (in addition to DC-SIGN.Fc and DC-SIGNR.Fc) was shown tobe able to capture DV2 (FIG. 16 a). To confirm the specificity of theinteraction between DLVR1/CLEC5A and DV, complexes wereimmunoprecipitated with protein A sepharose beads and then probed withan anti-DV envelope (anti-E) monoclonal antibody (mAb). E protein wasdetected in the immunoprecipitates of DC-SIGN.Fc and DLVR1/CLEC5A.Fc,confirming that DLVR1/CLEC5A interacts with the dengue virion (FIG. 16b). However, while the binding of DC-SIGN to DV is Ca⁺⁺-dependent, EDTA(a Ca⁺⁺ chelator) had no effect on the DLVR1/CLEC5A-DV interaction (FIG.1 c). Furthermore, transfection of 293T cells with DC-SIGN andDLVR1/CLEC5A resulted in increased binding of biotinylated DV to thecells (FIG. 1 d).

There are two conserved N-linked glycosylation sites at Asn-67 andAsn-153 of E protein (Pokidysheva, E. et al., Cell 124:485-93 (2006)),and the attached glycans (with terminal fucose and mannose) have beenimplicated in cellular attachment and viral entry (Modis, Y. et al., J.Virol. 79:1223-1231 (2005)). To investigate the participation of glycansin the association of DLVR1/CLEC5A with DV, virions were incubated withfucose, mannose, or mannan; where the last two sugars are the ligandsfor DC-SIGN (Mitchell et al., J. Biol. Chem. 276: 28939-28945 (2006)).As expected, mannose and mannan caused dose-dependent inhibition of theDC-SIGN-DV interaction (FIG. 16 e), while the binding of DLVR1/CLEC5A toDV was significantly reduced in the presence of fucose (p<0.0001) and,to a lesser extent, mannan (p=0.0005) (FIG. 16 e). Pre-treatment of DVwith PNGaseF also substantially reduced the DLVR1/CLEC5A-DV interaction(FIG. 16 f), suggesting that glycans present on the viral E proteins areessential for binding. Heat treatment or dithiothreitol (DTT) were alsofound to abolish the DLVR1/CLEC5A-DV interaction (FIG. 16 f), suggestingthat the correct topographical distribution of glycans on the denguevirion is important.

Example 17 Interaction of DC-SIGN and DLVR1/CLEC5A with Dengue Virus

FIG. 17 shows the expression patterns of DC-SIGN and DLVR1/CLEC5A inhuman PBMCs. Freshly isolated PBMCs were double stained withPE-conjugated antibodies to CD markers (BD PharMingen) andFITC-conjugated anti-DC-SIGNmAb, according to embodiments shown in FIG.17 a or FITC-conjugated anti-DLVR1/CLEC5A mAb (R&D Systems) according toembodiments shown in FIG. 17 b. CD marker-positive cells were gated todetermine the expression of DC-SIGN and DLVR1/CLEC5A (dashed lines).Shaded areas represent isotype controls.

DC-SIGN, which is expressed on DCs and macrophages (FIG. 17 a), containsthree motifs in its cytoplasmic tail that are believed to be involved ineither endocytosis or intracellular trafficking (Lozach et al., J. Biol.Chem. 2005, 280, 23698 23708). In contrast, DLVR1/CLEC5A was originallyidentified as DAP12-associated molecule expressed exclusively onmonocytes and macrophages (FIG. 17 b), although its ligand(s) andbiological functions remain to be determined (Bakker et al., Proc. Natl.Acad. Sci. USA 1999, 96, 9792 9796).

Example 18 DLVR1/CLEC5A is Essential for DV-Induced DAP12Phosphorylation, But Not For Dengue Virus Replication

According to embodiments illustrated in FIG. 18, DLVR1/CLEC5A isessential for DV-induced DAP12 phosphorylation, but not for DVreplication. Specifically, in FIG. 18 a, DV-induced DAP12phosphorylation (2 h p.i.) in human macrophages was determined usingantibodies to phosphotyrosine and DAP12 on western blots. FIG. 18 billustrates the kinetics of DAP12 phosphorylation induced by DV andUV-inactivated DV (UV-DV).

FIG. 17 c illustrates the ability of shRNAs to knock down the expressionof DLVR1/CLEC5A and DC-SIGN and to inhibit DV-mediated (m.o.i.=5) DAP12phosphorylation. FIG. 18 d shows the effects of shRNAs on DV entry andreplication in macrophages as determined by flow cytometry. FIG. 18 eillustrates the effects of anti-DLVR1/CLEC5A mAb, anti-DC-SIGN mAb, andmouse IgG (50 μg/ml) on the expression of nonstructural protein NS3(red; Cy3 labeled) examined by confocal microscope (Tassaneetrithep, B.et al., J Exp Med 197: 823-29 (2003)). Cells were counter stained withHoechst 33342 (blue). In both FIG. 18 d and FIG. 18 e, macrophages wereinfected with DV (m.o.i.=5) to determine NS3 expression at 48 hpost-infection. FIG. 18 f shows the effect of shRNAs on the DV titers ofinfected macrophages.

Infection of macrophages with DV was found to induce DAP12phosphorylation in a dose-dependent manner (FIG. 18 a). DAP12phosphorylation peaked at 12 h post-infection (p.i.) and persisted forat least 48 h in the presence of live DV, while UV-inactivated denguevirus (UV-DV) triggered only limited DAP12 phosphorylation that lastedfor just 12 h (FIG. 18 b), indicating that DAP12 phosphorylation isindependent of DV replication during the first 2-6 h of infection.Knockdown of DLVR1/CLEC5A (using the shRNA pLL3.7/DLVR1/CLEC5A), but notthat of DC-SIGN (by pLL3.7/DC-SIGN), caused a substantial reduction inDAP12 phosphorylation (FIG. 18 c), suggesting that DV-triggered DAP12phosphorylation is mediated via DLVR1/CLEC5A.

It is known that DC-SIGN participates in the infection of DCs by DV(Navarro-Sanchez et al., EMBO Rep. 4:723-28 (2003); Tassaneetrithep etal., J. Exp. Med. 197:823-829 (2003)). Therefore, DLVR1/CLEC5A wastested as to whether it is involved in DV entry into macrophages bymonitoring the expression of DV nonstructural protein 3 (NS3), which isexpressed when DV replicates in macrophages. In contrast to DC-SIGN,knockdown of DLVR1/CLEC5A by shRNA (FIG. 18 d) or blocking of theDLVR1/CLEC5A-DV interaction with anti-DLVR1/CLEC5A Ab (FIG. 18 e) didnot inhibit NS3 expression in macrophages as examined by flow cytometryand confocal microscopy, respectively. The shRNA pLL3.7/DLVR1/CLEC5Aalso failed to suppress the release of dengue virions into thesupernatant of infected macrophages, as determined by the plaque-formingassay (FIG. 18 f). These results indicate that, while DC-SIGN mediatesDV infection and replication, the interaction of DV with DLVR1/CLEC5Atriggers cell signaling.

Example 19 Inhibition of DLVR1/CLEC5A-Dengue Virus InteractionSuppresses Inflammatory Response by Infected Macrophages WithoutAffecting Viral Clearance Response

To determine whether DLVR1/CLEC5A is involved in DV-inducedinflammation, the secretion of inflammatory cytokines by macrophages,following infection with DV, was examined.

According to embodiments illustrated in FIG. 19, DLVR1/CLEC5A iscritical for DV-mediated TNF-α, but not IFN-α, secretion. Morespecifically, FIG. 19 a illustrates the dose-dependency of DV and UV-DVinduced TNF-α secretion by macrophages measured by ELISA at 6 h and 12 hp.i. FIG. 19 b illustrates the kinetics of TNF-α expression after DVinfection (m.o.i.=5). FIG. 19 c illustrates the effects of DLVR1/CLEC5Aand DC-SIGN shRNAs on the secretion of TNF-α, IL-6, MIP1-α, IL-8, IP-10,and IFN-α from DV-infected macrophages (m.o.i.=5). In FIG. 19 d,knockdown experiments with receptor specific shRNAs illustrating thatDV-induced IFN-α secretion is via the TLR7-MyD88 pathway and TNF-αsecretion is via DLVR1/CLEC5A-TLR7-MyD88 pathway. FIG. 19 e illustratesantagonistic anti-DLVR1/CLEC5A mAbs inhibiting TNF-α secretion inresponse to DV serotypes 1-4 is inhibited (see Table 3).

TABLE 3 Characteristics of anti-human DLVR1/CLEC5A mAbs. Iso-Antagonistic Clone type ELISA W.B. FACS DV1 DV2 DV3 DV4 Agonistic 3E12C1IgG1 + + + ** ** ** * − 3E12A2 IgG1 + + + ** ** ** * − 3E12G9 IgG1 + + +− ** ** − − 8H8F5 IgG1 + + + * *** ** * − 2H11 IgM + − + − − − − +9B12H4 IgM + − + *** *** − − − “*” - p < 0.05; “**” - p < 0.01; “***” -p < 0.001. Antibodies (10 μg/sample) that can suppress DV-induced TNF-αsecretion from human macrophages. Two-tailed Student's t-tests wereperformed and data were compared with each appropriate isotype controlantibody. An antibody that can trigger TNF-α secretion from humanmacrophages (compared with isotype control) is defined as agonistic. Thespecificity was confirmed by abolition of TNF-α secretion usingpLL3.7/DLVR1/CLEC5A-shRNA (pLL3.7 vector and pLL3.7/DC-SIGN had noeffect on antibody mediated TNF-α secretion. ELISA, Enzyme-Linked ImmunoSorbent Assay; WB, western blotting; FACS, Fluorescence Activated CellSorting. Antibodies are azide-free, sterile-filtered, and with endotoxinlevel less than 0.1EU per micrograms.M.R. mAb (anti-mannose receptor mAb; mIgG1) and murine IgM (mIgM) wereused as negative controls. In both FIG. 19 d and FIG. 19 e, macrophageswere infected with DV (m.o.i.=5) and harvested at 36 h p.i. for cytokineassays. Data are expressed as mean±s.d. of three independent experiments(using material from at least three different donors). Two-tailed,Student's t-tests were performed. “ND” represents not detected.

At 6 h p.i., dose-dependent secretion of TNF-α was detected, wheresimilar levels of cytokine were secreted by macrophages infected witheither DV or UV-DV (FIG. 19 a, left panel). However, at 12 h p.i., TNF-αsecretion was further increased by DV, but not by UV-DV (FIG. 19 a,right panel). Over a 48 h time course, TNF-α secretion continuallyincreased for macrophages infected with DV, while at 24 h-48 hpost-infection with UV-DV this cytokine was barely detectable (FIG. 19b). These data are in accord with the kinetics of DAP12 phosphorylation(FIG. 18 b), suggesting that DV-mediated TNF-α secretion is related toDAP12 activation. It was also observed that knockdown of DLVR1/CLEC5Asuppressed the release of TNF-α, IL-6, IL-8, MIP1-α, and IP-10 byDV-infected macrophages to a greater extent than knockdown of DC-SIGN(FIG. 19 c). However, while pLL3.7/DC-SIGN mildly suppressed IFN-αsecretion (p=0.048), pLL3.7/DLVR1/CLEC5A had no effect on IFN-α (FIG. 19c).

To further understand the DV-activated signaling pathways leading tocytokine secretion, macrophages were transfected with shRNAs to knockdown DLVR1/CLEC5A, DC-SIGN, TLR4, TLR7 or MyD88, prior to DV infection.The data obtained indicate that DV-induced IFN-α secretion occurs viathe TLR7—MyD88 pathway (p=0.0016), while TNF-α secretion is mediatedthrough both DLVR1/CLEC5A (p=0.003) and TLR7-MyD88 (p=0.013) (FIG. 19d). A panel of anti-DLVR1/CLEC5A mAbs was generated, with differentialantagonistic effects on the four serotypes of DV (see Table 3 above) asdetermined by inhibition of TNF-α secretion from DV-infected macrophages(FIG. 3 e). These data indicate that, although different epitopes ofDLVR1/CLEC5A appear to mediate the individual interactions, antibodiesthat inhibit the DLVR1/CLEC5A-DV interaction can suppress theinflammatory response by macrophages infected with the relevant DVserotype(s). The differential antagonistic effects of anti-DLVR1/CLEC5AmAbs might relate to the fact that each DV serotype binds to distinctepitope of DLVR1/CLEC5A, and anti-DLVR1/CLEC5A mAb can inhibit thebinding of specific DV serotype whose binding site overlaps with that ofanti-DLVR1/CLEC5A mAb.

Example 20 Antibody-Dependent Enhancement (ADE) Mediated IFN-a Secretionis Independent of DLVR1/CLEC5A

It has been demonstrated previously that non-neutralizing anti-DV Abspromote DV entry into target cells via FcR receptors and, thereby,enhance cytokine release (Halstead et al., J. Exp. Med. 146:201-217(1977); Goncalvez et al., Proc Natl Acad Sci USA 104:9422-9427 (2007)),a phenomenon termed antibody-dependent enhancement (ADE) of infection.For example, anti-prM and anti-E mAb have been shown to induce thiseffect in vitro (Huang et al., J Immuno 176:2825-2832 (2006)). Here,investigation was conducted regarding whether blockade of theDLVR1/CLEC5A-DV interaction can inhibit ADE.

As according to embodiments illustrated in FIG. 20, DLVR1/CLEC5A iscritical for ADE-mediated secretion of TNF-α but not IFN-α. Morespecifically, in FIG. 20 a macrophages were infected with DV (m.o.i.=5),DV/anti-E or DV/anti-prM immunocomplexes (ADE) for 36 h, followed by thedetection of DV replication by anti-NS3 mAb. Macrophages from 10individuals were infected with DV2 (FIG. 20 b) or DV/anti-prM orDV/anti-E complexes (FIG. 20 c), in the presence of antagonisticanti-DLVR1/CLEC5A mAb (1 μg; clone 9B12H4) or isotype control. TNF-α andIFN-α secretion were determined by ELISA. Two-tailed, Student's t-testswere performed.

Primary human macrophages were infected with DV alone or withanti-prM/DV or anti-E/DV immunocomplexes, in the presence ofanti-DLVR1/CLEC5A mAb (or isotype control) for 36 h. Anti-prM/DV andanti-E/DV immunocomplexes (ADE) were found to increase the expression ofNS3 (FIG. 20 a) and the levels of TNF-α and IFN-α secretion as comparedto DV alone (FIGS. 20 b and 20 c). However, while the anti-DLVR1/CLEC5AmAb significantly inhibited TNF-α release from macrophages infected withDV, anti-prM/DV and anti-E/DV immunocomplexes (FIG. 20 c), IFN-αsecretion was not affected, suggesting that ADE-mediated IFN-α secretionis independent of DLVR1/CLEC5A (as noted above for DV-induced IFN-αproduction).

Example 21 Involvement of DLVR1/CLEC5A in Dengue-Virus Induced VascularLeakage

The hallmarks of DHF and DSS are plasma leakage together withsubcutaneous and vital organ hemorrhaging. These symptoms are caused bythe numerous soluble mediators and cytokines released by immune cells toincrease vascular permeability (Green et al., Curr. Opin. Infect. Dis.19:429-436 (2006)). To determine whether DLVR1/CLEC5A is involved inDV-induced vascular leakage, monolayers of human dermal microvascularendothelial cells (HMEC-1) were used in a permeability assay (Carr etal., J. Med. Virol. 69:521-528 (2003)).

According to embodiments illustrated in FIG. 21, antagonisticanti-DLVR1/CLEC5A mAbs rescue the permeabilization of endothelial cellmonolayers by the supernatants of DV infected macrophages. Morespecifically, FIG. 21 a illustrates changes over time in thepermeability of HMEC-1 monolayers determined by measurement of HRPpassage following incubation with supernatants from macrophages infectedwith DV or DV/anti-prM complexes (ADE). TNF-α levels in the supernatantswere measured by ELISA. As illustrated in FIG. 21 b, the inhibitoryeffects of TNFR2.Fc (5 μg/ml) and anti-DLVR1/CLEC5A (clone 9B12H4, 5μg/ml) on permeabilization of endothelial monolayers were determined.Data are expressed as mean±s.d. of three independent experiments.Two-tailed, Student's t-tests were performed.

Supernatants from macrophages infected with DV or anti-prM/DVimmunocomplexes were found to induce permeability in HMEC-1 monolayers;where the immunocomplexes (ADE) produced a more significant effect thanDV alone, during the first 36 h-48 h of infection (FIG. 21 a, left).Interestingly, the changes in permeability do not correlate with TNF-αlevels in the supernatants (FIG. 21 a, right). Furthermore, whileneutralization of TNF-α by recombinant TNFR2.Fc was able to partiallyinhibit the induction of permeability triggered by DV or anti-prM/DV(p<0.05) (FIG. 21 b), the anti-DLVR1/CLEC5A mAb was more effective inthis respect (FIG. 21 b). It was observed that anti-DLVR1/CLEC5A blockedthe secretion of other inflammatory cytokines by macrophages, inaddition to TNF-α (FIG. 19 c), might explain this phenomenon.

Example 22 Dose-Dependent Interaction Between Antagonistic Mabs andDengue-Virus Induced TNF-α Secretion

Further investigation was conducted on whether blockade of the DV-CLEC5Ainteraction can rescue mice from DV-induced lethality in vivo. Accordingto embodiments illustrated in FIG. 22, the interaction of mDLVR1/CLEC5Aand DV (FIG. 22 a) and expression pattern of mDLVR1/CLEC5A in murinecells (FIGS. 22 b and 22 c) is shown. More specifically, FIG. 22 a showsthe interaction of DV (5×10⁶ PFU) with human and murine DLVR1/CLEC5A.Fc(1 μg) determined by ELISA. F4/80 and CD marker-positive cells weregated to determine the expression of mDLVR1/CLEC5A in murine splenocytes(FIG. 22 b), murine bone marrow (BM)-derived macrophage and the murinemacrophage cell line Raw264.7 (FIG. 22 c).

It was found that murine DLVR1/CLEC5A (mDLVR1/CLEC5A) binds DV with asimilar affinity as human DLVR1/CLEC5A (FIG. 22 a), and mDLVR1/CLEC5A isexpressed on myeloid lineages (CD11b+, F4/80+), bone marrow derivedmacrophages, and murine macrophage-like Raw264.7 cells (FIGS. 22 b and22 c).

Example 23 Blockade of mDLVR1/CLEC5A-DV Interaction SuppressesDV-Induced TNF-α Secretion from Raw264.7 Cells

According to embodiments illustrated in FIG. 23, blockade ofmDLVR1/CLEC5A-DV interaction suppresses DV-induced TNF-α secretion fromRaw264.7 cells. More specifically, FIG. 23 a shows that human DC-SIGNincreased DV binding to the murine macrophage cell line Raw264.7 andenhanced the stimulatory effect of DV infection. TNF-α release wasdetermined by ELISA. FIG. 23 b shows the identification of antagonisticmAbs to murine DLVR1/CLEC5A. Raw264.7 cells stably expressing humanDC-SIGN were incubated with DV2 (PL046; m.o.i.=30) in the presence ofmAbs. TNF-α levels in supernatants (at 48 h p.i.) were determined byELISA. FIG. 23 c shows that anti-mDLVR1/CLEC5A mAbs (clone: 3D2H6 and10D7H3) inhibit DV2-(NGC-N; m.o.i.=30) induced TNF-α release in adose-dependent manner. mIgG1 acts as an isotype-matched negativecontrol.

DV stimulated Raw264.7 cells stably expressing human DC-SIGN(Raw264.7/DC-SIGN) to secrete TNF-α (FIG. 23 a), and blockade ofmDLVR1/CLEC5A-DV interaction by antagonistic mAbs (Table 4) abolishedDV-induced TNF-α secretion by Raw264.7/DC-SIGN cells in a dose-dependentmanner (FIGS. 23 b and 23 c).

TABLE 4 Characteristics of anti-murine DLVR1/CLEC5A mAbs. Antagonisticeffect Clone Isotype ELISA FACS DV2 DV2 (NGC-N) 3D2H6 IgG1 + + * **9D8E9 IgG1 + ND * ND 9D9F9 IgG1 + ND − ND 1A10F9 IgG1 + ND − ND 10D7G4IgG1 + ND − ND 10D7H3 IgG1 + + * * “*” - p < 0.05; “**” - p < 0.01;“ND” - not done. Antibodies that suppress DV-induced TNF-α secretionfrom murine macrophages cell line RAW264.7/DC-SIGN. Two-tailed Student'st-tests were performed and data were compared with each appropriateisotype control antibody. ELISA, Enzyme-Linked Immuno Sorbent Assay;FACS, Fluorescence Activated Cell Sorting. Antibodies are azide-free,sterile-filtered, and with endotoxin level less than 0.1EU permicrograms.

Example 24 Dengue Virus (NGC-N) Induces Lethality in STAT1^(−/−) Mice

According to embodiments illustrated in FIG. 24, STAT1^(−/−) mice(n=5/group) were challenged with DV2/PL046 or DV2/NGC-N strain (i.p. andi.c. routes) at a range of doses (from 102 to 105 PFU) for 4 weeks. Dataare exhibited as Kaplan-Meier survival curves.

IFN-α functions to inhibit viral replication in both infected anduninfected cells, and IFN-mediated responses to DV infection involveboth the STAT1-dependent (essential in the control of viral replication)and STAT1-independent (essential for resolution of infection) pathways(Shresta, et al. J. Immunol. 175:3946-3954 (2005)). Although wild typemice were resistant to DV infection, STAT1-deficient (STAT^(−/−))(Durbin, et al., Cell. 84:443-450 (1996)) mice were sensitive to DV2-9(strain New Guinea C-N) induced lethality (FIG. 24).

Example 25 Potential Therapeutic Effects of Antagonistic mAbs AgainstDLVR1/CLEC5A

Further testing was conducted on the potential therapeutic effects ofthe antagonistic mAbs on STAT1^(−/−) mice. According to embodimentsillustrated in FIG. 25, anti-DLVR1/CLEC5A mAbs prevent DV-inducedvascular leakage and lethality in STAT1-deficient mice. Morespecifically, in FIG. 25 a, the mAb 3D2H6 raised against murineDLVR1/CLEC5A inhibits the subcutaneous and intestinal hemorrhaging ofDV-challenged STAT1^(−/−) mice. In FIG. 25 b, plasma leakage into thevital organs of DV-challenged STAT-1^(−/−) mice was reduced by mAbsagainst DLVR1/CLEC5A (3D2H6 and 10D7H3), as determined by Evans blueassay. FIG. 25 c illustrates the quantification of vascular permeabilityby extraction of Evan blue from organs. Data are expressed as themean±s.d. of three independent experiments: *p<0.05; **p<0.01;***p<0.001 (Student's t test). FIG. 25 d illustrates the serum levels ofTNF-α and IP-10 (n=8; upper and middle) and virus titers (n=4; lower)for DV-challenged STAT11-1 mice at day 7 p.i. in the presence or absenceof anti-DLVR1/CLEC5A mAbs or TNFR2.Fc. Two-tail, Student's t-tests wereperformed. FIG. 25 e illustrates the survival curve of STAT1-deficientmice challenged with DV2 (strain New Guinea C-N, 1×105 PFU/mouse i.p.plus i.c. routes) in the presence of antagonistic anti-murineDLVR1/CLEC5A mAbs or TNFR2.Fc. Data were collected from four independentexperiments (17 mice in each group) and exhibited as Kaplan-Meiersurvival curves with log rank test. p values for significant differencebetween treatment of DLVR1/CLEC5A mAb and mouse IgG are indicated.

DV-challenged STAT1-mice exhibited ruffled fur and mild paralysis inaddition to subcutaneous and intestinal hemorrhaging at 8 days p.i.(FIG. 25 a), and all died within 7-14 days of infection (FIG. 25 e).Five doses of Abs (100 μg/mouse, i.p.) or TNFR2.Fc (100 μg/mouse, i.p.)were administered on days 0, 1, 3, 5, and 7 p.i. At 9 days p.i., leakageof Evans blue into the kidney, liver, stomach, small intestine, largeintestine, and spleen of DV-challenged mice was significantly reduced inmice treated with anti-mDLVR1/CLEC5A mAbs compared to controls (FIGS. 25b and 25 c). Anti-mDLVR1/CLEC5A mAbs also effectively lowered the serumlevels of TNF-α and IP-10 (FIG. 25 d, upper and middle), withoutsuppressing viral replication, at day 7 p.i. (FIG. 25 d, lower) andprotected mice from lethality at day 14 p.i. (70% protection rate). Theoverall survival rate of anti-mDLVR1/CLEC5A-treated mice is 48% asobserved at day 21 p.i. (FIG. 25 e), with DV being cleared from serum ofsurviving mice at day 23 p.i. (data not shown). Thus, blockade of theDLVR1/CLEC5A-DV interaction appears to prevent the DV-associatedcomplications of hemorrhaging and plasma leakage, as well as suppressingthe macrophage inflammatory response, without impairing virus clearanceby the adaptive immune response. In contrast, TNFR2.Fc neither reducedvascular permeability (FIG. 25 c) nor protected mice from lethality(FIG. 5 e), even though it effectively lowered the serum level of TNF-α(FIG. 25 d).

Example 26 DLVR1/CLEC5A is Involved in JEV-Mediated DAP12Phosphorylation and TNF-α Secretion from Human Macrophages

Like with DV, JEV follows a similar virus infection response pattern,which is believed to be the same or similar in all flaviviruses. Asillustrated in FIG. 26 a, interaction of DLVR1/CLEC5A.Fc (1 μg), withJEV and DV (5×10⁶ PFU), respectively, were determined by ELISA. DVinteracts with human DLVR1/CLEC5A (187 amino acid in length) SEQ ID NO:72, but not the alternatively spliced form sDLVR1/CLEC5A (aa 43-65 isdeleted). SEQ ID NO: 73. In contrast, JEV only interacts withsDLVR1/CLEC5A, but not full length DLVR1/CLEC5A. As illustrated in FIG.26 b, DV induces DAP12 phosphorylation (at 2 h p.i.) in humanmacrophages is shown. DAP12 in DV-infected macrophages were precipitatedby anti-DAP12 mAb, blotted to nitrocellulose paper after fractionationon SDS-PAGE, followed by incubation with antibodies againstphosphotyrosine and DAP12, respectively. JEV-induced DAP12phosphorylation (m.o.i.=5) is inhibited by pLL3.7/DLVR1/CLEC5A. Asillustrated in FIG. 26 c, kinetics of TNF-α secretion from humanmacrophages in response to JEV infection (left) are shown. JEV-inducedTNF-α secretion is inhibited by pLL3.5/DLVR1/CLEC5A mAb (right). Dataare expressed as the mean±s.d. of three independent experiments.

Example 27 Variable Heavy and Light Chain Sequence for mAB 3E12A2

The variable heavy chain sequence for mAb 3E12A2 is shown below (SEQ ID NO: 60):  1CAGGTGCAGC TCGAGGAGTC AGGACCTGAG CTGGTGAAAC CCGGGGCATC AGTGAAGCTG TCCTGCAAGG CTTCTGGCTA CACCTTCACTGAGTATATTA 101TACACTGGGT AAAGCAGAGG TCTGGACAGG GTCTTGAGTG GATTGGGTGG TTTTACCCTG GAAGTGGTAG TATAAAGTAC AATGAGAAATTCAAGGACAA 201GGCCACATTG ACTGCGGACA AATCCTCCAG CACAGTCTAT ATGGAGCTTA GTGGATTGAC ATCTGAAGAC TCTGCGGTCT ATITCTGTGCAAGACACGAT 301GGTTACTCCT ACTTTGACTA CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCAGCCAAA ACGACACCCC CATCTGTCTA TCCACTGGCCCCTGGATCTG 401CTGCCCAAAC TAACTCCATG GTGACCCTGG GATGCCTGGT CAAGGGCTAT TTCCCTGAGC CAGTGACAGT GACCTGGAAC TCTGGATCCCTGTCCAGCGG 501TGTGCACACC TTCCCAGCTG TCCTGCAGTC TGACCTCTAC ACTCTGAGCA GCTCAGTGAC TGTCCCCTCC AGCACCTGGC CCAGCGAGACCGTCACCTGC 601AACGTTGCCC ACCCGGCCAG CAGCACCAAG GTGGACAAGA AANTTGTGCC CAGGGATTGT ACTAGTAAGC CTThe variable light chain sequence for mAb 3E12A2 is shown below (SEQ ID NO: 61):  1CCAGTTCCGA GCTCGTGACA CAGTCTCCAT CCTCCCTGGC TGTGTCAGCA GGAGAGAAGG TCACTATGAG CTGCAAATCC AGTCAGAGTCTGCTCAACAG 101TAGAACCCGA AAGAACTACT TGGCTTGGTA CCAGCAGAAA CCAGGGCAGT CTCCTAAACT GCTGATCTAC TGGGCATCCA CTAGGGAATCTGGGGTCCCT 201GATCGCTTCA CAGGCAGTGG ATCTGGGACA GATTTCACTC TCACCATCAG CAGTGTGCAG GCTGAAGACC TGGCAGTTTA TTACTGCAAGCAATCTTATA 301ATCTGTACAC GTTCGGAGGG GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT CTTCCCACCA TCCAGTGAGCAGTTAACATC 401TGGAGGTGCC TCAGTCGTGT GCTTCTTGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT GATGGCAGTG AACGACAAAATGGCGTCCTG 501AACAGTTGGA CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG ACGAGTATGA ACGACATAACAGCTATACCT 601GTGAGGCCAC TCACAAGACA TCAACITCAC CCATTGTCAA GAGCTTCAAC AGGAATGAGT GTTAATTCTA GACGGCGC

Example 28 Variable Heavy and Light Chain Sequence for mAb 3E12G9

The variable heavy chain sequence for mAb 3E12G9 is shown below (SEQ ID NO: 62):  1CAGGTGCAGC TCGAGCAGTC AGGACCTGAG CTGGTGAAAC CCGGGGCATC AGTGAAGCTG TCCTGCAAGG CTTCTGGCTA CACCTTCACTGAGTATATTA 101TACACTGGGT AAAGCAGAGG TCTGGACAGG GTCTTGAGTG GATTGGGTGG TTTTACCCTG GAAGTGGTAG TATAAAGTAC AATGAGAAATTCAAGGACAA 201GGCCACATTG ACTGCGGACA AATCCTCCAG CACAGTCTAT ATGGAGCITA GTGGATTGAC ATCTGAAGAC TCTGCGGTCT ATTTCTGTGCAAGACACGAT 301GGTTACTCCT ACTTTGACTA CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCAGCCAAA ACGACACCCC CATCIGTCTA TCCACTGGCCCCTGGATCTG 401CTGCCCAAAC TAACTCCATG GTGACCCTGG GATGCCTGGT CAAGGGCTAT TTCCCTGAGC CAGTGACAGT GACCTGGAAC TCTGGATCCCTGTCCAGCGG 501TGTGCACACC TGTCCAGCGG TCCTGCAGTC TGACCTCTAC ACTCTGACCA GCTCAGTGAC TGTCCCCTCC AGCACCTGGC CCAGCGAGACCGTCACCTGC 601AACGTTGCCC ACCCGGCCAG CAGCACCAAG GTGGACAAGA AAATTGTGCC CAGGGATTGT ACTAGTAAGC CTThe variable light chain sequence for mAb 3E12G9 is shown below (SEQ ID NO: 63):  1CCAGTTCCGA GCTCGTGACA CAGTCTCCAT CCTCCCTGGC TGTGTCAGCA GGAGAGAAGG TCACTATGAG CrGCAAATCC AGTCAGAGTCTGCTCAACAG 101TAGAACCCGA AAGAACTACT TGGCTTGGTA CCAGCAGAAA CCAGGGCAGT CTCCTAAACT GCTGATCTAC TGGGCATCCA CTAGGGAATCTGGGGTCCCT 201GATCGCTTCA CAGGCAGTGG ATCTGGGACA GATTTCACTC TCACCATCAG CAGTGTGCAG GCTGAAGACC TGGCAGTTTA TTACTGCAAGCAATCTTATA 301ATCTGTACAC GTTCGGAGGG GGGACCAAGC TGGAAATAAA ACGGGCTGAT GCTGCACCAA CTGTATCCAT CTTCCCACCA TCCAGTGAGCAGTTAACATC 401TGGAGGTGCC TCAGTCGTGT GCTTCITGAA CAACTTCTAC CCCAAAGACA TCAATGTCAA GTGGAAGATT GATGGCAGTG AACGACAAAATGGCGTCCTG 501AACAGTTGGA CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG ACGAGTATGA ACGACATAACAGCTATACCT 601GTGAGGCCAC TCACAAGACA TCAACTFCAC CCAT1GTCAA GAGCTTCAAC AGGAATGAGT GTTAATTCTA GACGGCGC

Example 29 Variable Heavy and Light Chain Sequence for mAb 8H8F5

The variable heavy chain sequence for mAb 8H8F5 is shown below (SEQ ID NO: 64):  1GAGGTGAAGC TCGAGGAGTC TGGACGAGGC TTAGTGCAGC CTGGAGGGTC CCGGAAACTC TCCTGTGCAG CCTCTGGATT CACTTTCAGTACCTCTGGAA 101TGCACTGGGT TCGTCAGGCT CCAGAGAAGG GGCTGGAGTG GGTCGCATAC ATTAGTAGTG GCAGCACTAC CATCTACCAT GCAGACACAGTGAAGGGCCG 201ATTCACCATC TCCAGAGACA ATCCCAAGAA CACCCTGTTC CTGCAAATGA CCAGTCTAAG GTCTGAGGAC ACGGCCATGT ATTACTGTGCAAGATCGGGT 301CAGTTTGGTA ACTACTTTGA CTACTGGGGC CAAGGCACCA CTCTCACAGT CTCCTCAGCC AAAACGACAC CCCCATCTGT CTATCCACTGCCCCTGGATC 401TGCTGCCCAA ACTAACTCCA TGGTGACCCT GGGATGCCTG GTCAAGGGCT ATTFCCCTGA GCCAGTGACA GTGACCTGGA ACTCTGGATCCCTGTCCAGC 501GGTGTGCACA CCTTCCCAGC TGTCCTGCAG TCTGACCTCT ACACTCTGAG CAGCTCAGTG ACTGTCCCCT CCAGCACCTG GCCCAGCGAGACCGTCACCT 601GCAACGTTGC CCACCCGGCC AGCAGCACCA AGGTGGACAA GAAAATTGTG CCCAGGGATT GTACTAGTAA GCCTThe variable light chain sequence for mAb 8H8F5 is shown below (SEQ ID NO: 65):  1CCAGATGTGA GCTCGTCATG ACCCAGTCTC CAAAATTCCT GCTTGTATCA GCAGGAGACA GGGTTACCCT AACCTGCAAG GCCAGTCAGAGTGTGAATAA 101TGATGTATAT TGGTACCAAC AGGAGCCAGG TCAGTCTCCT AAACTGCTGA TATACTATGC ATCCAATCGC TACACTGGAG TCCCTGATCGCTTCACTGGC 201AGTGGATATG GGACGGATTT CACTTTCACC ATCAGCACTG TGCAGTCTGA AGACCTGGCA GTTTATTTCT GTCAGCACGA TTATAGCTCTCCGTACACGT 301TCGGAGGGGG GACCAAGCTG GAAATAAAAC GGGCTGATGC TGCACCAACT GTATCCATCT TCCCACCATC CAGTGAGCAG TTAACATCTGGAGGTGCCTC 401AGTCGTGTGC TTCTTGAACA ACTTCTACCC CAAAGACATC AATGTCAAGT GGAAGATTGA TGGCAGTGAA CGACAAAATG GCGTCCTGAACAGTTGGACT 501GATCAGGACA GCAAAGACAG CACCTACAGC ATGAGCAGCA CCCTCACGTT GACCAAGGAC GAGTATGAAC GACATAACAG CTATACCTGTGAGGCCACTC 601ACAAGACATC AACTTCACCC ATTGTCAAGA GCTTCAACAG GAATGAGTGT TAATTCTAGA CGGCGC

Example 30 Comparison of Variable Heavy and Light Chain SequenceAlignment for mAb 8H8F5, 3E12A2, and 3E12G9

The comparison of the variable heavy chain alignment for mAb 8H8F5,3E12A2, and 3E12G9 is shown below (SEQ ID NO: 66, 67, and 68,respectively):

      10      20    30 40 508H8F5 VH4 (-----ESGRG LVQPGGSRKL SCAASGFTFS TSGMHWVRQA PEKGLEWVAY3E12A2 VH1 QVQLEESGPE LVKPGASVKL SCKASGYTFT EYIIHWVKQR SGQGLEWIGW3E12G9 VH9 QVOLEOSGPE LVKPGASVKL SCKASGYTFT EYIIHWVKQR SGQGLEWIGW      60      70    80    90          1008H8F5 VH4 (ISSGSTTIYH ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TAMYYCARSG3E12A2 VH1 FYPGSGSIKY NEKFKDKATL TADKSSSTVY MELSGLTSED SAVYFCARHD3E12G9 VH9 FYPGSGSIKY NEKFKDKATL TADKSSSTVY MELSGLTSED SAVYFCARHD 110(SEQ ID NO: 66) 8H8F5 VH4 (QFGNYFDYWG QGTTLTVSS (SEQ ID NO: 67)3E12A2 VH1 GYS-YFDYWG QGTTLTV-- (SEQ ID NO: 68)3E12G9 VH9 GYS-YFDYWG QGTTLTV--

The comparison of the variable light chain alignment for mAb 8H8F5,3E12A2, and 3E12G9 is shown below (SEQ ID NO: 69, 70, and 71,respectively):

       10     20    30 40 508H8F5 VL6 (MTQSPKFLLV SAGDRVTLTC KASQSVNND- -----VYWYQ QEPGQSPKLL3E12A2 VL6 --QSPSSLAV SAGEKVTMSC KSSQSLLNSR TRKNYLAWYQ QKPGQSPKLL3E12G9 VL2 --QSPSSLAV SAGEKVTMSC KSSQSLLNSR TRKNYLAWYQ QKPGQSPKLL       60     70    80    90          1008H8F5 VL6 (IYYASNRYTG VPDRFTGSGY GTDFTFTIST VQSEDLAVYF CQHDYSSPYT3E12A2 VL6 IYWASTRESG VPDRFTGSGS GTDFTLTISS VQAEDLAVYY CKQSYN-LYT3E12G9 VL2 IYWASTRESG VPDRFTGSGS GTDFTLTISS VQAEDLAVYY CKQSYN-LYT  110(SEQ ID NO: 69) 8H8F5 VL6 (FGGGTKLEIK R (SEQ ID NO: 70)3E12A2 VL6 FGGGTKLEIK - (SEQ ID NO: 71) 3E12G9 VL2 FGGGTKLEIK -

DLVR1/CLEC5A interacts with the dengue virion directly and, thereby,leads to DAP12 phosphorylation. Blockade of DLVR1/CLEC5A-DV interactionsuppresses the secretion of proinflammatory cytokines without affectinginterferon-α release. Moreover, anti-DLVR1/CLEC5A monoclonal antibodiesinhibit DV-induced plasma leakage, as well as subcutaneous and vitalorgan hemorrhaging, and reduce the incidence of DV infection by ˜50% inSTAT1-deficient mice. The results suggest that DV-triggered cytokinerelease from macrophages involves both DLVR1/CLEC5A and TLR7 pathways,while the blockade of DLVR1/CLEC5A-DV interactions attenuatesinflammation without preventing the clearance of virus. However, theblockade of TLR7 (or MyD88) receptors inhibits secretion of bothpro-inflammation cytokines, as well as viral clearance cytokines, whichultimately prevents the viral clearance as well as inflammation. Thus,effective treatment of dengue virus, as well as other flaviviruses suchas Japanese encephamyelitis virus, requires attenuation of viral bindingto DLVR1/CLEC5A, but not TLR7 or MyD88 receptors. Consequently, blockingof binding by dengue virus with anti-DLVR1/CLEC5A antibodies provides atherapy for severe dengue disease progression in DHF/DSS patients.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the hybridomas deposited,since the deposited embodiments are intended to illustrate only certainaspects of the invention and any antibodies that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that they represent. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

1-54. (canceled)
 55. A method for treating a viral infection, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition including an anti-human DVLR1/CLEC5A receptorantibody and a pharmaceutically acceptable carrier.
 56. The method ofclaim 55, wherein the composition contains a humanized antibody of theanti-human DVLR1/CLEC5A antibody.
 57. The method of claim 55, whereinthe anti-human DVLR1/CLEC5A antibody includes a heavy chain variablesequence (V_(H)) of SEQ ID NO:66 and a light chain variable sequence(V_(L)) of SEQ ID NO:69.
 58. The method of claim 55, wherein theanti-human DVLR1/CLEC5A antibody includes a V_(H) sequence of SEQ IDNO:67 and V_(L) sequence of SEQ ID NO:70.
 59. The method of claim 55,wherein the anti-human DVLR1/CLEC5A antibody includes a V_(H) sequenceof SEQ ID NO:68 and V_(L) sequence of SEQ ID NO:71.
 60. The method ofclaim 55, wherein the viral infection is caused by a hepatitis virus ora flavivirus.
 61. The method of claim 60, wherein the viral infection iscaused by a flavivirus selected from the group consisting of denguevirus, Japanese encephalomyelitis virus, and West Nile virus.